Macrophage Transfer to HSCs Assigns Residence in Bone Marrow

Hematopoietic stem cells (HSCs), residing at the apex of the hematopoietic hierarchy, are a rare and heterogeneous cell population. Although HSC subsets with various repopulation capacities and lineage bias have been identified, there is no available information about whether each HSC has an equal chance of being mobilized or whether there are specific pools of mobilizable or non-mobilizable HSCs in bone marrow (BM). Here, we identify a BM resident HSC subset based on the expression of the macrophage marker F4/80. Flow cytometry analysis revealed that F4/80 was expressed on 75% of HSCs (Lin- Sca1+cKit+CD150+CD34-; hereafter referred to as HSCM) in the BM. In competitive transplantation assays, HSCM exhibited significantly lower engraftment potential, but no lineage bias, compared to F4/80- HSCs (HSCM: 19.7±4.8%; F4/80- HSC: 37.1±4.5%, P<0.05). We next analyzed the ability of HSCM and F4/80- HSCs to be mobilized after G-CSF treatment and found that F4/80- HSCs, but not HSCM, were exclusively mobilized (F4/80- HSC: 5042±912.5; HSCM: 6.6±6.6 per mL blood, P<0.001). Similar selective mobilization was observed after administration of AMD3100 or macrophage depletion using clodronate liposomes. To confirm this observation in a genetic model, we crossed transgenic mice expressing Cre recombinase knocked into the Cd169 locus, a marker of BM macrophages (MΦ), with ROSA26-loxP-stop-loxP-tdTomato (CD169/Tomato). We found CD169/Tomato labelled a large fraction of HSCM (31.7±8.4%), in contrast to F4/80- HSCs which were largely unlabelled (2.1±1.2%). Remarkably, CD169/Tomato+ HSCs were not mobilized into the circulation after G-CSF treatment (CD169/Tomato- HSC: 4573±1416; CD169/Tomato+ HSC: 90.6±90.6 per mL blood, P<0.01). Although HSCs are reported to express Csf1r, the expression of macrophage markers on HSCs (F4/80 and CD169) was unexpected. To ascertain the expression origin, we transplanted CD169/Tomato+ and CD169/Tomato- sorted HSCs into lethally irradiated CD45.1 congenic mice. If HSCs expressed CD169, we would expect that donor HSC tomato expression would be retained. However, to our surprise, we found that the proportion of Tomato+ HSCs was higher in the CD169/Tomato- group, suggesting that F4/80 and CD169 expression may be acquired, rather than expressed within HSCs. Indeed, co-culture of TdTomato+ BM MΦ with GFP+ HSCs consistently revealed the acquisition of tdTomato (GFP+ TdTomato+: 9.9±1.9%). Transwell experiments revealed that cell contact was important as GFP+ cells did not show any TdTomato expression in the absence of direct cell contact (GFP+TdTomato+: 0%). To investigate the mechanism of HSC retention, we analyzed CXCR4 expression on HSCM and F4/80- HSC subsets. These analyses revealed that CXCR4 was expressed at higher expression levels on HSCM compared to F4/80- HSCs (HSCM: 79.4±9.1% CXCR4+;F4/80- HSC: 45.6±8.5% CXCR4+, P<0.001). In accordance with their increased CXCR4 expression, HSCM exhibited a 4.7-fold increase in pERK1/2 after CXCL12 stimulation, while CXCL12 only triggered a modest increase (1.5-fold) in pERK1/2 in F4/80- HSCs, suggesting that HSCM have a higher signaling response to CXCL12, and thus exhibit enhanced BM retention. To further confirm that the cell transfer occurred in vivo, we used xenografted mice in which human HSCs were transplanted into NOG mice. We found a significant presence of murine F4/80 on human CD34+ HSCs in xenografted huNOG mice, confirming the F4/80 transfer from host MΦ into human HSCs in vivo. To investigate further the mechanism of cell transfer, we carried out co-culture experiments of GFP+ lineage-depleted BM cells with TdTomato+ MΦ in the presence of inhibitors known to prevent cell transfer through membrane and cytoskeleton remodeling. Of all conditions tested, only the presence of the GAP junction inhibitor carbenoxolone (CBX) could inhibit the transfer of TdTomato from MΦ to GFP+ cells (control: 21.4±3.0%; CBX: 12.6±2.1%, P<0.001). Furthermore, treatment of wild-type mice with CBX led to a marked suppression of F4/80 transfer in vivo, similar to that observed in vitro, suggesting that GAP junctions are responsible for the direct transfer of cellular content from BM MΦ to HSCs. Our results thus identify a novel mechanism by which macrophages can assign HSC residence in BM. Manipulation of macrophage-mediated transfer may enhance the mobilizable HSC pool and provide a new method to improve HSC yields after mobilization. Disclosures Frenette: Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties; Pfizer: Consultancy.

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Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽

10.1182/blood-2007-03-081448 ◽

2007 ◽

Vol 110 (7) ◽

pp. 2276-2285 ◽

Cited By ~ 45

Author(s):

Maria De La Luz Sierra ◽

Paola Gasperini ◽

Peter J. McCormick ◽

Jinfang Zhu ◽

Giovanna Tosato

Keyword(s):

Bone Marrow ◽

Dna Sequences ◽

Peripheral Blood ◽

Cell Function ◽

Myeloid Cell ◽

Cxcr4 Expression ◽

Negative Regulator ◽

Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

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Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma

Blood ◽

10.1182/blood.2020005399 ◽

2020 ◽

Vol 136 (23) ◽

pp. 2607-2619 ◽

Cited By ~ 6

Author(s):

Karin Golan ◽

Abhishek K. Singh ◽

Orit Kollet ◽

Mayla Bertagna ◽

Mark J. Althoff ◽

...

Keyword(s):

Bone Marrow ◽

Connexin 43 ◽

Purinergic Receptor ◽

Cell Contact ◽

Hematopoietic Stem ◽

Mitochondrial Transfer ◽

Stem And Progenitor Cells ◽

Mitochondria Transfer ◽

Bone Marrow Regeneration

Abstract The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

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Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Blood ◽

10.1182/blood.v116.21.404.404 ◽

2010 ◽

Vol 116 (21) ◽

pp. 404-404 ◽

Cited By ~ 1

Author(s):

Heather A Himburg ◽

Pamela Daher ◽

J. Lauren Russell ◽

Phuong Doan ◽

Mamle Quarmyne ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Signaling Pathways ◽

Fold Increase ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Hscs ◽

Necessary And Sufficient

Abstract Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

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Targeted Clearance of Human Hematopoietic Stem Cell Niches Via Inhibition of SCF Signaling Using Monoclonal Antibody SR-1

Blood ◽

10.1182/blood.v116.21.74.74 ◽

2010 ◽

Vol 116 (21) ◽

pp. 74-74

Author(s):

Agnieszka Czechowicz ◽

Rashmi Bhardwaj ◽

Christopher Y. Park ◽

Irving L. Weissman

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Cord Blood ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Conditioning Regimens ◽

Human Hscs

Abstract Abstract 74 Hematopoietic stem cells (HSCs) are used therapeutically in bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) to correct hematolymphoid abnormalities. Upon intravenous transplantation, HSCs can home to specialized bone marrow niches, self-renew and differentiate and thus generate a new, complete hematolymphoid system. Unfortunately BMT has had limited applications, due to the risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy, that are deemed necessary for HSC engraftment. Elimination of these toxic conditioning regimens could expand the potential applications of BMT to include many non-malignant hematologic disorders, a wide variety of autoimmune disorders such as diabetes and multiple sclerosis, as well as in the facilitation of organ transplantation. We have previously shown that one important barrier to HSC engraftment is availability of HSC niche space. In the absence of pre-transplant conditioning, >99% of HSC niches are occupied with host HSCs and thus donor HSC engraftment under these conditions is minimal. We have shown in mouse models, that elimination of host mouse HSCs using anti-mouse-ckit monoclonal antibody ACK2 allows for >90% donor HSC engraftment with minimal toxicity in immunodeficient animals, which is sufficient to cure most hematolymphoid disorders. To examine the effects of inhibition of ckit-signaling in human HSCs, we obtained and created various monoclonal antibodies to human ckit and verified that anti-human-ckit monoclonal antibody SR-1 uniquely inhibits SCF binding. To examine the dependence of human HSC on ckit-signaling we cultured purified human bone marrow HSC and umbilical cord blood HSC (CD34+CD38-CD90+CD45RA-Lin-) in the presence of StemSpan media supplemented with human cytokines SCF, TPO, Flt-3, IL-3, IL-6 and either 10ug/ml of SR-1 or 4F7 (an anti-human-ckit clone that did not inhibit SCF-binding). Here we show that SR-1 uniquely inhibited both human bone marrow and cord blood HSC proliferation in vitro. Interestingly, SR-1 did not induce human HSC cell death via apoptosis, as addition of Z-VAD-FMK caspase inhibitor did not abrogate the effects of SR-1. Additionally, the lack of PI+ and Annexin V+ populations day 3 and 7 days post addition of SR-1 failed to provide evidence of HSC death. However, careful examination revealed a shift in the differentiation profile of HSC cultured in the presence of SR-1. Whereas 4F7 did not change the in vitro output of human HSC, cord blood HSC cultured in the presence of SR-1 showed increased propensity to give rise to Glycophorin A+ and CD41+ cells (RBC and platelets respectively), and decreased output of CD14, CD13, and CD33 cells (macrophages/myeloid cells). To examine the depleting capability of SR-1 in vivo, robust human-mouse hematopoietic chimeras were generated. Newborn immune deficient NOD/SCID/IL-2Rγnull (NOG) mice received 100cGy radiation and subsequently were transplanted intravenously via facial vein with ∼1000 purified human cord blood HSC. Mice with >15% human CD45+ chimerism in the peripheral blood at 12 wks post-transplant were selected for further experimentation. Bone marrow aspirates were obtained from these animals and assayed for human chimerism by FACS. Average pre-treatment baseline level of total human bone marrow engraftment (%hCD45) was 58.6%, whereas myeloid chimerism (%hCD13/33) was 25.4%. The mice were then treated intravenously with 500ug of SR-1 every other day for 1 week. Human total and myeloid engraftment 8 weeks post treatment decreased by 92.4% and 96.9% respectively, most likely due to depletion of human HSCs that maintain these populations. The remaining persistent human cells in these animals were primarily composed of mature, long-lived B and T-cells that do not need to be regenerated by HSC and are therefore unaffected by this therapy. In summary, we have shown that human HSCs depend on SCF for proliferation, and inhibition of SCF-signaling via anti-ckit monoclonal antibody SR-1 resulted in non-toxic, specific, in vitro and in vivo HSC depletion. Through this method we hope to deplete host HSCs in humans, thereby increasing available human HSC niches for engraftment and providing effective yet mild conditioning prior to transplantation. This work could enable efficient transplantation of HSC that cannot cause GvH, resulting in a potential curative therapy for almost any hematologic or immunologic disease. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Non-Invasive Multimodal/Multi-Scale Imaging of Bone Marrow Microenvironment Reveals Structural and Functional Heterogeneity in Different Bone Marrow Compartments: Functional Implications On Mouse and Human Hematopoietic Stem Cell Niches in Health and Disease

Blood ◽

10.1182/blood.v120.21.25.25 ◽

2012 ◽

Vol 120 (21) ◽

pp. 25-25

Author(s):

Francois Lassailly ◽

Katie Foster ◽

Lourdes Lopez-Onieva ◽

Erin Currie ◽

Dominique Bonnet

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endochondral Ossification ◽

Bone Marrow Microenvironment ◽

Hematopoietic Stem ◽

Non Invasive ◽

Health And Disease ◽

Human Hscs ◽

Stem Cell Niches

Abstract Abstract 25 Introduction: Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) microenvironments called stem cell niches (HSCNs). Two types of HSCNs have been reported, involving osteoblasts (osteoblastic niche) and blood sinusoids (vascular niche). Various hematopoietic and non-hematopoietic cell types are contributing to the regulation of HSCs and HSCNs, however, the bone marrow microenvironment has not yet been precisely characterized and the fine localization, composition and regulation of the niches remain highly controversial. Intravital microscopy of the calvarium (IVMC) is the only non-invasive option for high resolution imaging of bone marrow HSCNs. However it is unclear if IVMC provides data representative of all BM compartments. Indeed it has been shown that endochondral ossification, the process used for long bones growth, is required for the formation of HSCNs. By contrast, the calvarium is a flat bone which develops through intra-membranous ossifications, therefore questioning the presence of HSCNs in this bone (Chan CK et al, Nature 2009). Methods: Combined confocal and multiphoton excitation intravital microscopy was used for multiparametric observation of live bone marrow in intact bones after in-vivo contrasting procedures. Bioluminescence imaging was used to quantify the systemic trafficking and proliferation of murine HSCs and human leukemia. Flow cytometry and histology were used to sort specific populations of cells, analyze the frequency of mouse and human stem cells or leukemic cells at steady state or after transplantation, quantify perfusion efficiency and hypoxia and cross-validate in-vivo imaging procedures. Results: Thanks to advanced imaging modalities we realized a thorough study of potential niche markers and HSCs distribution at homeostasis and during hematopoietic reconstitution in calavia, epiphyses and diaphyses. We report important heterogeneity between these compartments in terms of bone remodelling activity (BRA) and blood vessel fraction (BVF). Although BVF was surprisingly high in any compartment, including in very close proximity to any endosteal surface, we found that compartments displaying the highest BVF and BRA were preferentially seeded and engrafted following mouse and human HSC transplantation. Unexpectedly, the macro-anatomical distribution of mouse and human HSCs at steady state is homogeneous across the skeleton and independent of these 2 markers. These data suggest the existence of “reconstituting niches” which would be distinct from “homeostatic niches”. Importantly, this study provides the first evidence that both types of niches are fully functional in the calvarium, including for mouse and human HSCs, indicating that endochondral ossification is dispensable for adult HSCNs. The model is currently being exploited to analyze leukemia/microenvironment interactions in live bone marrow. Conclusions: This study confirms and extends our recent statement considering the critical need for multimodal imaging (Lassailly F et al, Blood 2010). Further more, it demonstrates that combination of different imaging modalities for in-vivo and ex-vivo analysis is a powerful strategy allowing to shed a new light on the structure of the bone marrow microenvironment and improve our understanding of stem cells/niches interactions in health and disease. Disclosures: No relevant conflicts of interest to declare.

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CD8+TCR- Bone Marrow Facilitating Cells Prime Migration and Enhance Homing of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.2412.2412 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2412-2412

Author(s):

Yujie Wen ◽

Yiming Huang ◽

Thomas O Miller ◽

Mariusz Z Ratajczak ◽

Suzanne T Ildstad

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Cxcr4 Expression ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Lower Chamber ◽

Hematopoietic Niche ◽

Marrow Cells

Abstract CD8+TCR- bone marrow facilitating cells (FCs) facilitate engraftment of hematopoietic stem cells (HSCs) in both allogeneic and syngeneic recipients. We recently reported that co-administration of FCs with HSCs established chimerism and induced tolerance to renal allografts without graft-versus-host disease and engraftment syndrome in HLA-mismatched living donor renal transplant recipients. Homing of HSCs to the bone marrow niche is believed to be a crucial prerequisite for engraftment. Therefore, we evaluated whether FCs enhance functional HSC homing and retention in the hematopoietic niche using the in vivo syngeneic homing model followed by colony-forming cell (CFC) assay. B6 mice were conditioned with a supralethal dose (1200 cGy) of total body irradiation (TBI) and transplanted with 75,000 B6 FCs, 25,000 B6 HSCs or FCs plus HSCs 24 hours after TBI. At 18 hours after transplantation, bone marrow cells were harvested from the recipient’s femurs and tibias and placed in CFC assay using methylcellulose-based media. Bone marrow cells harvested from mice transplanted with FCs without HSCs or those receiving conditioning alone did not generate colonies, confirming that FCs themselves do not have repopulating capacity in vivo and post-TBI bone marrow was free of recipient HSCs under the supralethal dose of TBI. FCs significantly enhanced the efficiency of HSC homing as reflected by the bone marrow cells harvested from the mice transplanted with HSCs and FCs formed significantly higher numbers of colonies compared to that of the mice transplanted with HSCs alone (24.7 ± 2.9 vs. 15.6 ± 2.7; p < 0.05). The chemokine receptor CXCR4 plays a pivotal role in HSC homing. We therefore determined whether the effect of FCs on HSC homing was mediated by FCs increasing CXCR4 expression in HSC. CXCR4 expression in HSCs was measured by flow cytometry 18 hours after co-culture of HSCs and FCs in vitro. Incubation of FCs with HSCs did not lead to increased CXCR4 expression on HSCs compared with HSCs cultured alone. We next tested whether FCs enhance the responsiveness of HSCs to a low concentration of stromal cell-derived factor 1 (SDF-1, also known as CXCL12) gradient in Transwell chemotaxis assays in vitro. 500 HSCs were placed in the upper chamber and tested for migration of cells to the lower chamber of the Transwell in response to a 30 ng/ml SDF-1 gradient for 3 hours. The cells harvested from the lower chamber were placed in CFC assay. The migrated cells harvested from the lower chamber containing FCs formed 6-fold more colonies than HSCs obtained from the lower chamber that did not contain FC (22.3 ± 3.9 vs. 4.0 ± 1.2; p < 0.05). Furthermore, we found that small factions of total FCs were CXCL12-producing cells (2% of total FCs) and CD169+ cells (5% of total FCs). The migration ability of FCs was characterized by following patterns of homing after transplantation of 300,000 CellTracker Green labeled FCs or 300,000 FCs bearing different congenic marker. In 8,000,000 bone marrow cells harvested from femurs and tibias of the transplanted mice 18 hours post-transplantation, we detected 100 to 300 of transplanted CellTracker Green labeled FCs. On the 10 serial bone sections from femurs and tibias of the transplanted mice, we enumerated 15 to 20 of CellTracker Green labeled FCs. These data suggested that FCs might home to bone marrow within 18 hours post-transplantation. In summary, our results suggest that FCs enhance functional HSC homing and retention in the hematopoietic niche in vivo. This is not mediated by increased CXCR4 expression in HSCs. FCs also prime HSC migration to a low concentration SDF-1 gradient in vitro, possibly through production of priming factors. Disclosures Ildstad: Regenerex, LLC: Other.

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CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽

10.1182/blood.v87.10.4136.bloodjournal87104136 ◽

1996 ◽

Vol 87 (10) ◽

pp. 4136-4142 ◽

Cited By ~ 113

Author(s):

I Kawashima ◽

ED Zanjani ◽

G Almaida-Porada ◽

AW Flake ◽

H Zeng ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Marrow Cell ◽

In Utero ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Show Evidence ◽

Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

Blood ◽

10.1182/blood-2010-04-277897 ◽

2011 ◽

Vol 117 (2) ◽

pp. 451-458 ◽

Cited By ~ 58

Author(s):

Mamiko Noda ◽

Yoshiki Omatsu ◽

Tatsuki Sugiyama ◽

Shinya Oishi ◽

Nobutaka Fujii ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Nk Cell ◽

Cell Development ◽

Hematopoietic Stem ◽

Adult Mice ◽

Multipotent Progenitors ◽

Chemokine Signaling ◽

Chemokine Ligand

Abstract Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

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