Joji Fujisaki

Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche

Stem cells reside in a specialized, regulatory environment termed the niche that dictates how they generate, maintain and repair tissues. We have previously documented that transplanted haematopoietic stem and progenitor cell populations localize to subdomains of bone-marrow microvessels where the chemokine CXCL12 is particularly abundant. Using a combination of high-resolution confocal microscopy and two-photon video imaging of individual haematopoietic cells in the calvarium bone marrow of living mice over time, we examine the relationship of haematopoietic stem and progenitor cells to blood vessels, osteoblasts and endosteal surface as they home and engraft in irradiated and c-Kit-receptor-deficient recipient mice. Osteoblasts were enmeshed in microvessels and relative positioning of stem/progenitor cells within this complex tissue was nonrandom and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different haematopoietic cell subsets localized to distinct locations according to the stage of differentiation. When physiological challenges drove either engraftment or expansion, bone-marrow stem/progenitor cells assumed positions in close proximity to bone and osteoblasts. Our analysis permits observing in real time, at a single cell level, processes that previously have been studied only by their long-term outcome at the organismal level.

In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche.

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 (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg 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.

In vivo imaging of Treg cells providing immune privilegeto the haematopoietic stem-cell niche

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 (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg 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.

Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

In vivo imaging of T reg cells providing immune privilege to the haematopoietic stem-cell niche

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 (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg 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.

In vivo imaging of T reg cells providing immune privilegeto the haematopoietic stem-cell niche

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 (Treg) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with Treg cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. Treg cells seem to participate in creating a localized zone where HSPCs reside and where Treg 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.

Persistence of donor-derived protein in host myeloid cells after induced rejection of engrafted allogeneic bone marrow cells

OBJECTIVE In recipients of allogeneic hematopoietic stem cell transplantation to treat hematologic malignancies, we have unexpectedly observed anti-tumor effects in association with donor cell rejection in both mice and humans. Host-type CD8 T cells were shown to be required for these anti-tumor effects in the murine model. Because sustained host CD8 T-cell activation was observed in the murine bone marrow following the disappearance of donor chimerism in the peripheral blood, we hypothesized that donor antigen presentation in the bone marrow might be prolonged. MATERIALS AND METHODS To assess this hypothesis, we established mixed chimerism with green fluorescent protein (GFP)-positive allogeneic bone marrow cells, induced rejection of the donor cells by giving recipient leukocyte infusions, and utilized in vivo microscopy to follow GFP-positive cells. RESULTS After peripheral donor leukocytes disappeared, GFP persisted within host myeloid cells surrounding the blood vessels in the bone marrow, suggesting that the host myeloid cells captured donor-derived GFP protein. CONCLUSIONS Because the host-vs-graft reaction promotes induction of anti-tumor responses in this model, this retention of donor-derived protein may play a role in the efficacy of recipient leukocyte infusions as an anti-tumor therapy.