Project description:Disseminated tumor cells in the bone marrow environment are the main cause of systemic metastasis after curative treatment for major solid tumors. However, the detailed biological processes of tumor biology in bone marrow have not been well defined in a real-time manner, because of a lack of a proper in vivo experimental model thereof. In this study, we established intravital imaging models of the bone marrow environment to enable real-time observation of cancer cells in the bone marrow. Using these novel imaging models of intact bone marrow and transplanted bone marrow of mice, respectively, via two-photon microscopy, we could first successfully track and analyze both the distribution and the phenotype of cancer cells in bone marrow of live mouse. Therefore, these novel in vivo imaging models for the bone marrow would provide a valuable tool to identify the biologic processes of cancer cells in a real-time manner in a live animal model.
Project description:In vivo pathways of natural retinoid metabolism and elimination have not been well characterized in primary myeloid cells, even though retinoids and retinoid receptors have been strongly implicated in regulating myeloid maturation. With the use of a upstream activation sequence-GFP reporter transgene and retrovirally expressed Gal4-retinoic acid receptor ? in primary mouse bone marrow cells, we identified 2 distinct enzymatic pathways used by mouse myeloid cells ex vivo to synthesize retinoic acid receptor ? ligands from free vitamin A metabolites (retinyl acetate, retinol, and retinal). Bulk Kit(+) bone marrow progenitor cells use diethylaminobenzaldehyde-sensitive enzymes, whereas bone marrow-derived macrophages use diethylaminobenzaldehyde-insensitive enzymes to synthesize natural retinoic acid receptor ?-activating retinoids (all-trans retinoic acid). Bone marrow-derived macrophages do not express the diethylaminobenzaldehyde-sensitive enzymes Aldh1a1, Aldh1a2, or Aldh1a3 but instead, express Aldh3b1, which we found is capable of diethylaminobenzaldehyde-insensitive synthesis of all trans-retinoic acid. However, under steady-state and stimulated conditions in vivo, diverse bone marrow cells and peritoneal macrophages showed no evidence of intracellular retinoic acid receptor ?-activating retinoids, despite expression of these enzymes and a vitamin A-sufficient diet, suggesting that the enzymatic conversion of retinal is not the rate-limiting step in the synthesis of intracellular retinoic acid receptor ?-activating retinoids in myeloid bone marrow cells and that retinoic acid receptor ? remains in an unliganded configuration during adult hematopoiesis.
Project description:The craniofacial skeleton is the foundation of most stomatological treatments, including prosthodontics and maxillofacial surgery. Although histologically similar to the appendicular skeleton, the craniofacial skeleton manifests many unique properties in response to external stimuli and signals. However, the mandibular or maxillary bone marrow mesenchyme, which is the intrinsic foundation of the functions of craniofacial skeleton, has not been well studied, and its homeostasis mechanism remains elusive. Osteoporosis is a systemic disease that affects all skeletons and is characterized by bone mass loss. Osteoporotic bone marrow mesenchymal stem cells (BMMSCs) exhibit disturbed homeostasis and distorted lineage commitment. Many reports have shown that microRNAs (miRNAs) play important roles in regulating MSCs homeostasis. Here, to obtain a better understanding of mandibular bone marrow MSCs homeostasis, we isolated and cultured mandible marrow MSCs from mouse mandibles. Using miR-705 mimics and an inhibitor, we demonstrated that miR-705 played a vital role in shifting the mandibular MSCs lineage commitment in vitro. Utilizing an osteoporosis mouse model, we demonstrated that MSCs from ovariectomized (OVX) mouse mandibular bone marrow exhibited impaired osteogenic and excessive adipogenic differentiation. miR-705 was found overexpressed in OVX mandibular MSCs. The knock down of miR-705 in vitro partially attenuated the differentiation disorder of the OVX mandibular MSCs by upregulating the expression of osteogenic marker genes but suppressing adipogenic genes. Taken together, our findings provide a better understanding of the homeostasis mechanism of mandibular BMMSCs and a novel potential therapeutic target for treating mandibular osteoporosis.
Project description:Irradiation-resistant natural killer (NK) cells in an F(1) recipient can reject parental bone marrow, and host NK cells can also prevent engraftment of allogeneic bone marrow. We show here that repopulating bone marrow cells in certain mouse strains expressed retinoic acid early inducible 1 proteins, which are ligands for the activating NKG2D NK cell receptor. Treatment with a neutralizing antibody to NKG2D prevented rejection of parental BALB/c bone marrow in (C57BL/6 x BALB/c) F(1) recipients and allowed engraftment of allogeneic BALB.B bone marrow in C57BL/6 recipients. Additionally, bone marrow from C57BL/6 mice transgenic for retinoic acid early inducible 1epsilon was rejected by syngeneic mice but was accepted after treatment with antibody to NKG2D. If other stem cells or tissues upregulate expression of NKG2D ligands after transplantation, NKG2D may contribute to graft rejection in immunocompetent hosts.
Project description:Mouse models of transplantation have been indispensable to the development of bone marrow transplantation (BMT). Their role in the generation of basic science knowledge is invaluable and is subject to discussion below. However, this article focuses on the direct role and relevance of mouse models towards the clinical development and advances in BMT and adoptive T-cell therapy for human diseases. The authors aim to present a thoughtful perspective on the pros and cons of mouse models while noting that despite imperfections these models are obligatory for the development of science-based medicine.
Project description:Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.
Project description:Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells responsible for myelin regeneration. Oligodendrocytes (OLGs) originate from OPCs and are the myelinating cells in the central nervous system (CNS). OLGs play an important role in the context of lesions in which myelin loss occurs. Even though many protocols for isolating OPCs have been published, their cellular yield remains a limit for clinical application. The protocol proposed here is novel and has practical value; in fact, OPCs can be generated from a source of autologous cells without gene manipulation. Our method represents a rapid, and high-efficiency differentiation protocol for generating mouse OLGs from bone marrow-derived cells using growth-factor defined media. With this protocol, it is possible to obtain mature OLGs in 7-8 weeks. Within 2-3 weeks from bone marrow (BM) isolation, after neurospheres formed, the cells differentiate into Nestin+ Sox2+ neural stem cells (NSCs), around 30 days. OPCs specific markers start to be expressed around day 38, followed by RIP+O4+ around day 42. CNPase+ mature OLGs are finally obtained around 7-8 weeks. Further, bone marrow-derived OPCs exhibited therapeutic effect in shiverer (Shi) mice, promoting myelin regeneration and reducing the tremor. Here, we propose a method by which OLGs can be generated starting from BM cells and have similar abilities to subventricular zone (SVZ)-derived cells. This protocol significantly decreases the timing and costs of the OLGs differentiation within 2 months of culture.
Project description:Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.
Project description:AIM:To determine if the small-molecule radioprotector GS-nitroxide, JP4-039, improved hematopoiesis in long-term bone marrow cultures (LTBMCs), explanted marrow from in vivo drug-treated C57BL/6NTac mice was maintained in JP4-039 for 25 weeks. Hematopoietic cell production and radiobiology of derived stromal cell lines was measured. MATERIALS AND METHODS:Groups of LTBMCs were established from mouse groups. Stromal cell lines were established from the adherent layer of JP4-039-treated and untreated control groups. RESULTS:LTBMCs maintained in JP4-039 exhibited increased production of total non-adherent and 7-day and 14-day hematopoietic colony-forming cells. Stromal cell lines derived from JP4-039-treated cultures were radioresistant in vitro, demonstrated a distinct squamous/epithelial morphology and overexpressed Nrf2, Ctgf, Lox, Tlr1, collagen 1a, Brd3, and Brd4. CONCLUSION:Chronic treatment of bone marrow cultures and derived stromal cell lines with JP4-039 was non-toxic, and conferred resistance to oxidative stress.
Project description:Basophils co-express Fc?RI? and CD49b, the ?-2 chain of integrin-type receptor VLA-2 (?2?1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (?-4, ?-5 and ?-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.