153 research outputs found
Identification of stromal cells in spleen which support myelopoiesis
Stromal cells in spleen organize tissue into red pulp, white pulp and marginal zone, and also interact with hematopoietic cells to regulate immune responses. This study has used phenotypic information of a previously described spleen stromal cell line called 5G3, which supports restricted hematopoiesis in vitro, to identify an equivalent stromal cell subset in vivo and to test its capacity to support hematopoiesis. Using stromal cell fractionation, phenotypic analysis, as well as cell growth and hematopoietic support assays, the Sca-1+gp38+Thy1.2+CD29+CD51+ fraction of spleen stroma has been identified as an equivalent stromal subset resembling the 5G3 cell counterpart. While heterogeneity may still exist within that subset, it has been shown to have superior hematopoietic support capacity compared with the 5G3 cell line, and all other spleen stromal cell fractions tested.This work was supported by project grants to HO from the Australian Research Council (#DP130101703) and the National Health and Medical Research Council of Australia (#585443). HL was supported by an Australian National University Postgraduate Scholarship
MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis
Background: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with
production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal
line produce two populations of cells, characterised as CD11b+CD11c+MHC-II− dendritic-like ‘L-DC’, and CD11b+CD11c+
MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of
these two subsets has not been clearly distinguished.
Results: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T
cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+
T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL10
production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the
proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDClike
cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene
expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype,
while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with
functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell
contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF
production by stroma.
Conclusion: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of
dendritic-like cells. These are here distinguished phenotypically and through gene expression differences.
While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the
cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through
Transwell co-cultures with cell production dependent on M-CSF.
Keywords: Hematopoiesis, Dendritic cell, Regulatory dendritic cells, Regulatory T cellsThis work was supported by project grant #585443 to HO from the National
Health and Medical Research Council of Australia. SP was supported by a
graduate scholarship from the Royal Thai Government. PP was supported by
an Australian National University Graduate Scholarship
Antigen presenting capacity of murine splenic myeloid cells
BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named ‘L-DC’ was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8(+) T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII(−) cells and unable to activate CD4(+) T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4(+) T cells or CD8(+) T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8(+) T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4(+) T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell
Development of Dendritic Cells from GM-CSF-/- Mice in vitro : GM-CSF Enhances Production and Survival of Cells
The production of dendritic cells (DC) from haemopoietic progenitors maintained in long
term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently
of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that
cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to
generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/-
and wild type mice were established and compared for cell production. GM-CSF-/- LTC
developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable
to wild type cultures. LTC maintained distinct populations of small and large cells, the latter
resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting
cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed
the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to
GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells
from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained
in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in
increasing the survival of mature DC
Role of SVEP1 in Stroma-Dependent Hematopoiesis In vitro
Study of the microenvironment that supports hematopoietic stem cell (HSC) development in vivo is very difficult involving small numbers of interacting cells which are usually not well defined. While much is known about HSC niches located within the bone marrow in terms of contributing cell types and signalling molecules, very little is known about equivalent niches within spleen. Extramedullary hematopoiesis in spleen contributes myeloid cells important in the mobilisation of an immune response. As a result, it is important to develop in vitro models to identify the cells which constitute HSC niches in spleen and to identify the regulatory molecules supporting myeloid cell development. Studies described here document a model system to study the maintenance and differentiation of HSC by splenic stromal cells in vitro. The splenic stromal lines 5G3 and 3B5 differ in hematopoietic support capacity. SVEP1 and IGF2 are molecules of interest specifically expressed by 5G3 stroma. Gene knockdown technology using shRNA plasmids has been used to reduce gene expression in 5G3 and to determine specific effects on myeloid cell development following co-culture with overlaid hematopoietic progenitors in vitro. Knockdown of Svep1 gave specific inhibition of a dendritic cell (DC) population described previously in spleen (L-DC). Knockdown of Igf2 resulted in loss of production of a minor subset of conventional (c) DC. SVEP1 is now considered a marker of mesenchymal stromal cells with osteogenic differentiative capacity reflective of perivascular stromal cells. The power of this in vitro model is evidenced by the fact that it has been used to define SVEP1 as a specific adhesion molecule that regulates the hematopoietic process dependent on stromal niche interaction. The identification of stromal cells and molecules that contribute to the hematopoietic process in spleen, brings us closer to the realm of therapeutically regulating hematopoiesis in vivo, and to inhibiting niches which support cancer stem cells
The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells
Escherichia coli’s heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and mye-loid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vi-tro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional deple-tion of CD11c+ DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells
Advancing a stem cell therapy for age-related macular degeneration
The retinal pigment epithelium (RPE) is a multifunctional monolayer located at the back of the eye required for the survival and function of the light-sensing photoreceptors. In Age-related Macular Degeneration (AMD), the loss of RPE cells leads to photoreceptor death and permanent blindness. RPE cell transplantation aims to halt or reverse vision loss by preventing the death of photoreceptor cells and is considered one of the most viable applications of stem cell therapy in the field of regenerative medicine. Proof-of-concept of RPE cell transplantation for treating retinal degenerative disease, such as AMD, has long been established in animal models and humans using primary RPE cells, while recent research has focused on the transplantation of RPE cells derived from human pluripotent stem cells (hPSC). Early results from clinical trials indicate that transplantation of hPSC-derived RPE cells is safe and can improve vision in AMD patients. Current hPSC-RPE cell production protocols used in clinical trials are nevertheless inefficient. Treatment of large numbers of AMD patients using stem cellderived products may be dependent on the ability to generate functional cells from multiple hPSC lines using robust and clinically-compliant methods. Transplantation outcomes may be improved by delivering RPE cells on a thin porous membrane for better integration into the retina, and by manipulation of the outcome through control of immune rejection and inflammatory responses
Efficient differentiation of human embryonic stem cells to retinal pigment epithelium under defined conditions
Transiently expressed CRISPR/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications
Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches
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