In Vivo Imaging of Hematopoietic Stem Cell Engraftment in the Mouse Tibia Bone

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1286-1286
Author(s):  
Seungbum Kim ◽  
Li Lin ◽  
Koji Hosaka ◽  
Edward W Scott
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Real Time ◽  
Cell Biology ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Tibia Bone ◽  
Cell Engraftment ◽  
Mouse Tibia

Abstract Abstract 1286 A fundamental question in stem cell biology is where stem cells reside and how stem cell niches control stem cell activity. Although the hematopoietic stem/progenitor cell (HSPC) is well characterized, our current knowledge of where and how transplanted HSPCs become engrafted is very limited. HSPC transplantation is now routinely done in clinics to correct a variety of bone marrow deficiencies. Considering its extensive use, understanding the HSPC engraftment process has now become critical if we are to improve the treatment strategies. A key to understanding HSPC engraftment is to be able to observe the process in vivo. We aimed to visualize fluorescent HSCs in the mouse tibia bone directly by grinding one side of the tibia until the bone was sufficiently thin for direct observation. By making a “window” into the tibia bone, we were able to observe the early engraftment process of a single HSC forming a colony in real time with high resolution. The Sca-1+, c-Kit+, Lin- (SKL) cells preferred to lodge and proliferate mainly on the osteoblastic niche. In contrast, further purified SKL, CD48-, CD150+ population (SLAM-SKL) was mostly observed in the perivascular niche. When mice were co-transplanted with DsRed+ SKL and GFP+ SLAM-SKL populations, SKL cells were 5–7 times more than SLAM-SKL cells in the bone marrow at Day 7. However, contribution of each population to the blood circulation at the same time was similar, which suggests that the SLAM-SKL cells engrafted in the perivascular niche can produce blood much quicker. We were not only able to observe the engrafted cells but also visualize donor derived cells circulating in the bone marrow in real time. Our study shows a novel technique to understand and highlight the dynamic process of the stem cell engraftment in complex microenvironment of the bone marrow. Disclosures: No relevant conflicts of interest to declare.

Specialized Bone Marrow Endothelium Defines Microdomains for Tumor and Stem Cell Engraftment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 663-663
Author(s):  
Dorothy A. Sipkins ◽  
Xunbin Wei ◽  
Juwell W. Wu ◽  
Terry K. Means ◽  
Andrew D. Luster ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Cells ◽  
Stem Cell Differentiation ◽  
Multiple Time ◽  
Normal Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Vascular Beds

Abstract The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation and regeneration, yet relatively little is known about the architecture of microenvironments that support malignant proliferation. Using dynamic in vivo confocal and multi-photon imaging, we show that the bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant SDF-1 in discrete, discontinuous areas that localize the homing and early engraftment of both leukemic and normal primitive hematopoietic cells. Real-time imaging of cell-cell interactions in SCID mice bone marrow was performed after injection of fluorescently-labeled leukemic and other malignant cell lines. Progressive scanning and optical sectioning through the marrow revealed the existence of unique, spatially-restricted vascular domains to which the majority of marrow-homing tumor cells rolled and arrested. Serial imaging of mice on days 3 – 14 demonstrated that leukemic (Nalm-6 pre-B ALL) extravasation and early proliferation were restricted to these vascular beds. To define the molecular basis of these homing interactions, in vivo labeling of key vascular cell adhesion molecules and chemokines using fluorescent antibodies was performed. We observed that while ICAM-1, VCAM-1, PECAM-1 and P-selectin were expressed diffusely throughout the marrow vasculature, the expression of E-selectin and the chemokine receptor CXCR4 ligand SDF-1 was distinctly limited to vessels that supported leukemic cell engraftment. In vivo co-localization experiments confirmed Nalm-6 binding was restricted to vascular beds expressing both E-selectin and SDF-1. In functional studies, disruption of E-selection had a modest effect on leukemic homing (<20% diminution), while pharmacologic blockade of CXCR4 decreased Nalm-6 binding to vessels by approximately 80%. To explore the normal function of this vascular niche, we next examined whether benign primitive hematopoietic cells might preferentially home to these same vascular microdomains. Fluorescently-labeled stem and progenitor cells (HSPC) isolated from donor balb/c mice were injected into recipient mice and imaging was performed at multiple time points. HSPC were found to adhere to the BM microvasculature in the same restricted domains. At 70 days post-injection, HSPC had extravasated, were persistent in these perivascular areas and had undergone cell division as assessed by dye dilution. Our findings show that these microdomains serve as vascular portals around which leukemic and hematopoietic stem cells engraft, suggesting that this molecularly distinct vasculature provides both a cancer and normal stem cell niche. Specialized vascular structures therefore appear to delineate a stem cell microenvironment that is exploited by malignancy.

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 857-857
Author(s):  
Gregor B. Adams ◽  
Ian R. Alley ◽  
Karissa T. Chabner ◽  
Ung-il Chung ◽  
Emily S. Marsters ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3478-3482 ◽  
Author(s):  
Xiuli Wang ◽  
Michael Rosol ◽  
Shundi Ge ◽  
Denise Peterson ◽  
George McNamara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Signal Intensity ◽  
Bioluminescence Imaging ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
In Vivo Bioluminescence Imaging

Abstract The standard approach to assess hematopoietic stem cell (HSC) engraftment in experimental bone marrow transplantation models relies on detection of donor hematopoietic cells in host bone marrow following death; this approach provides data from only a single time point after transplantation for each animal. In vivo bioluminescence imaging was therefore explored as a method to gain a dynamic, longitudinal profile of human HSC engraftment in a living xenogeneic model. Luciferase expression using a lentiviral vector allowed detection of distinctly different patterns of engraftment kinetics from human CD34+ and CD34+CD38- populations in the marrow NOD/SCID/β2mnull mice. Imaging showed an early peak (day 13) of engraftment from CD34+ cells followed by a rapid decline in signal. Engraftment from the more primitive CD34+CD38- population was relatively delayed but by day 36 increased to significantly higher levels than those from CD34+ cells (P &lt; .05). Signal intensity from CD34+CD38--engrafted mice continued to increase during more than 100 days of analysis. Flow cytometry analysis of bone marrow from mice after death demonstrated that levels of 1% donor cell engraftment could be readily detected by bioluminescence imaging; higher engraftment levels corresponded to higher image signal intensity. In vivo bioluminescence imaging provides a novel method to track the dynamics of engraftment of human HSC and progenitors in vivo. (Blood. 2003;102: 3478-3482)

Successful in vivo purging of CD34-containing peripheral blood harvests in mantle cell and indolent lymphoma: evidence for a role of both chemotherapy and rituximab infusion

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 864-869 ◽  
Author(s):  
Michele Magni ◽  
Massimo Di Nicola ◽  
Liliana Devizzi ◽  
Paola Matteucci ◽  
Fabrizio Lombardi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Follicular Lymphoma ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Bone Marrow Involvement ◽  
Hematopoietic Stem ◽  
Mantle Cell

Abstract Elimination of tumor cells (“purging”) from hematopoietic stem cell products is a major goal of bone marrow–supported high-dose cancer chemotherapy. We developed an in vivo purging method capable of providing tumor-free stem cell products from most patients with mantle cell or follicular lymphoma and bone marrow involvement. In a prospective study, 15 patients with CD20+ mantle cell or follicular lymphoma, bone marrow involvement, and polymerase chain reaction (PCR)–detectable molecular rearrangement received 2 cycles of intensive chemotherapy, each of which was followed by infusion of a growth factor and 2 doses of the anti-CD20 monoclonal antibody rituximab. The role of rituximab was established by comparison with 10 control patients prospectively treated with an identical chemotherapy regimen but no rituximab. The CD34+ cells harvested from the patients who received both chemotherapy and rituximab were PCR-negative in 93% of cases (versus 40% of controls;P = .007). Aside from providing PCR-negative harvests, the chemoimmunotherapy treatment produced complete clinical and molecular remission in all 14 evaluable patients, including all 6 with mantle cell lymphoma (versus 70% of controls). In vivo purging of hematopoietic progenitor cells can be successfully accomplished in most patients with CD20+ lymphoma, including mantle cell lymphoma. The results depended on the activity of both chemotherapy and rituximab infusion and provide the proof of principle that in vivo purging is feasible and possibly superior to currently available ex vivo techniques. The high short-term complete-response rate observed suggests the presence of a more-than-additive antilymphoma effect of the chemoimmunotherapy combination used.

scholarly journals Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment

Blood ◽  
2018 ◽  
Vol 132 (7) ◽  
pp. 735-749 ◽  
Author(s):  
Simranpreet Kaur ◽  
Liza J. Raggatt ◽  
Susan M. Millard ◽  
Andy C. Wu ◽  
Lena Batoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Key Points ◽  
Bone Marrow Engraftment

Key Points Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation. Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Correlation between Hematopoietic Stem Cell Engraftment Assay Results and Bone Marrow Biopsy, Flow Cytometry, and Cytogenetics Findings.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4961-4961
Author(s):  
Edward G. Weir ◽  
Kathleen Murphy ◽  
Denise Batista ◽  
Constance A. Griffin ◽  
Michael J. Borowitz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Host Cells ◽  
Fish Analysis ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Hematopoietic stem cell transplantation following induction chemotherapy is an increasingly successful therapeutic option for patients with leukemia or lymphoma. The use of molecular assays in post-transplant patients has become the standard in evaluating these patients for evidence of engraftment or early recurrence of disease. The detection of residual host cells in the bone marrow (BM) or peripheral blood (PB) following stem cell transplantation often influences subsequent clinical management. The aim of our study is to determine the extent of correlation between the results of PCR-based stem cell engraftment (SCE) assays and BM biopsy (BMBx), multiparameter flow cytometry (FC) and cytogenetics findings in patients who have undergone stem cell transplantation as therapy for hematolymphoid malignancies. We retrospectively reviewed the results of 1103 serial SCE assays performed at The Johns Hopkins Hospital, and 596 of these had temporally corresponding BMBx, FC and/or cytogenetic analysis. Concordance between the results of SCE analysis and those of the latter assays was defined as the detection of similar host/donor compositions. While some cases demonstrated clear discordance between the results, a subset showed an equivocal correlation due to the unclear significance of <5% host DNA by SCE analysis. Of 318 SCE assays with concurrent BMBx, 167(52%) showed concordant results, 104(33%) showed discordant results, and 47(15%) demonstrated an equivocal correlation. Of 221 SCE assays with concurrent FC, 111(50%) showed concordant results, 73(33%) showed discordant results, and 37(17%) demonstrated an equivocal correlation. Additionally, SCE assays were performed on concurrent, paired BM and PB specimens in 168 patients. Concordant results were identified in 141(84%) pairs. Of the remaining 27 pairs, host DNA was detected in the PB of 16 cases in which the BM showed either donor only DNA (6 cases) or <5% host DNA (10 cases). Four cases showed <5% host DNA in the PB and chimeric DNA in the BM. However, donor only DNA was detected in the PB in 7 cases that demonstrated a chimeric BM. Lastly, concurrent SCE analysis and XY FISH analysis was identified in 28 cases. Concordance between these two assays was observed in 24 (86%) cases, whereas one (3%) case was discordant and 3 (11%) cases showed an equivocal correlation. In conclusion, both BMBx and FC show similar but weak correlations to SCE analysis. In contrast, XY FISH analysis demonstrates a strong correlation to SCE analysis. Furthermore, SCE analyses performed on paired PB and BM specimens show an overall good correlation. However, our data suggest that in a subset of cases, SCE analysis performed on PB may detect residual host DNA that is not detectable by SCE analysis of BM, possibly due to the heterogeneity of the marrow composition.

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