Novel Role for Host-Derived Megakaryocytes In Facilitating Stem Cell Engraftment through Enhancement of Osteoblastic Niche Restoration Following Radioablation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 558-558
Author(s):  
Timothy S. Olson ◽  
Anna Caselli ◽  
Satoru Otsuru ◽  
Ted Hofmann ◽  
Edwin M. Horwitz
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Lymphocyte ◽  
Conflicts Of Interest ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Endosteal Surface ◽  
Post Transplant ◽  
Cell Engraftment ◽  
Donor Cells

Abstract Abstract 558 The osteoblastic niche is a critical site of engraftment following stem cell transplantation. We have previously demonstrated that the osteoblastic niche expands within the first 48 hours following marrow radioablation. This expansion is accompanied by relocalization of megakaryocytes from their homeostatic location within the central marrow space to the endosteal surface at sites of osteoblast expansion, suggesting that these relocalized megakaryocytes may play a key role in reconstitution of the niche. To determine whether megakaryocytes contribute to the radioablation-induced osteoblast expansion and consequently assist in facilitating engraftment, we have examined osteoblast expansion and stem cell engraftment in wildtype (WT) mice, thrombopoietin receptor (c-Mpl) deficient mice (mpl−/−) that have less than 20% of normal megakaryocyte numbers, and in mice treated with an anti-CD41 blocking antibody (MWREG30). BrdU incorporation and TUNEL assays demonstrated that megakaryocyte relocalization following radioablation occurs through active migration of viable megakaryocytes. mpl−/− mice or anti-CD41 treated WT mice developed less than 20% of the megakaryocyte endosteal migration seen in untreated WT mice 48 hours after radioablation (*P<0.001), and anti-CD41 treatment of irradiated mpl−/− mice reduced endosteal megakaryocytes to baseline pre-radiation numbers. Moreover, mpl−/− mice developed less than 50% of the osteoblast expansion seen in WT mice following irradiation; thus, abrogating megakaryocyte activity blocks critical signals required for osteoblast expansion. Bone marrow expression of IGF-1, a recognized osteoblast growth factor, increased 5-fold within 48 hours after radiation relative to pre-radiation levels in WT mice, but this IGF-1 spike is completely blocked by c-Mpl deficiency, suggesting that megakaryocytes may induce osteoblast expansion through a pathway in which c-Mpl signaling leads to IGF-1 expression. To test the functional significance of radiation-induced megakaryocyte migration and osteoblast expansion, we transplanted lethally irradiated WT or mpl−/− mice with or without anti-CD41 treatment using bone marrow from GFP-transgenic mice. Initial bone marrow engraftment of WT GFP+ donor cells within 24h of transplantation was significantly reduced (*P<0.01 for all groups) in anti-CD41 treated WT (51% of engraftment seen in untreated wildtype recipients), untreated mpl−/− (45%), and anti-CD41 treated mpl−/− (35%) recipients. Expansion of engrafted WT GFP+ donor cells at 3, 5 and 7 days post-transplant was also significantly reduced in untreated mpl−/− recipients (36%, 53%, 63% of untreated WT recipients at 3, 5, and 7 days, respectively, *P<0.05) and anti-CD41 treated mpl−/− recipients (25%, 33%, and 30% at 3, 5, and 7 days, respectively, *P<0.05), with prominent deficits specifically in the reconstitution of the B lymphocyte lineage. Bone marrow cellularity remained significantly reduced in anti-CD41 treated WT and untreated or anti-CD41 treated mpl−/− recipients by 35–45% relative to untreated WT recipients at least out to 3 weeks post-transplant (*P<0.01). Using competitive repopulation secondary transplantation assays performed with marrow harvested from primary recipients at 24h after primary transplantation, we showed that progenitor and short term hematopoietic stem cell (HSC) engraftment was significantly decreased in mpl−/− versus WT primary recipients (*P<0.05). Secondary transplant assays performed with marrow harvested from primary recipients 3 weeks after initial transplantation demonstrated that engraftment and expansion of long term-HSC (*P <0.05) and B cell reconstitution (*P<0.005) are significantly impaired in anti-CD41 treated mpl−/− versus untreated WT recipients. Taken together, our findings demonstrate that host megakaryocytes migrate to the endosteal surface following marrow radioablation where, through the enhancement of osteoblast niche expansion and potential osteoblast-independent effects, they play a pivotal role in facilitating efficient HSC engraftment following transplantation. Further understanding of these stem cell niche restoration pathways may reveal novel therapeutic targets to improve engraftment efficiency in the clinical setting. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 20-20
Author(s):  
James Bartram ◽  
Baobao (Annie) Song ◽  
Juying Xu ◽  
Nathan Salomonis ◽  
H. Leighton Grimes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Functional Decline ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

Impairment of Hematopoietic Stem Cell (HSC) Niche by G-CSF Is Associated with Rapid Mobilization of Serially Reconstituting HSC and Reduced Competitive Repopulation of Mobilized Bone Marrow

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1889-1889 ◽  
Author(s):  
Jean-Pierre Levesque ◽  
Valerie Barbier ◽  
Bianca Nowlan ◽  
Domenica McCarhty ◽  
Ingrid G Winkler
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Steady State ◽  
Conflicts Of Interest ◽  
Brdu Incorporation ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Donor Chimerism ◽  
Hsc Niche

Abstract Abstract 1889 We have previously shown that G-CSF administration impairs HSC niches in the mobilized bone marrow (BM). G-CSF causes rapid suppression (within 2 days) of endosteal osteoblasts and bone formation with concomitant down-regulation of Kit ligand, CXCL12 and angiopoietin-1. This effect is mediated by the depletion of specific populations of BM macrophages1. Considering the very rapid impairment of HSC niches in response to G-CSF, we hypothesized that 1) the most primitive HSC should also mobilize at this very early stage within the first 48 hours of G-CSF treatment, and 2) that down-regulation of HSC niche function should also alter the number or function of HSC remaining in the mobilized BM. To test this, 125μg/kg rhuG-CSF was injected twice daily to C57BL/6 mice; blood and BM harvested at days 2 and 5 of G-CSF treatment to be transplanted into congenic recipients in a long-term competitive repopulation assay (LT-CR). Transplantation of 25μL blood showed a gradual increase in the number of LT-CR cells mobilized in response to G-CSF as measured by donor chimerism in myeloid and B lineages at 16 weeks post-transplant. Expectedly repopulating units (RU) per mL blood progressively increased from 0.2 ± 0.0 (n=6) in steady-state to 2.9 ± 1.6 (n=9) and 82.6 ± 40.4 (n=9) at days 2 and 5 of G-CSF treatment respectively. At 16 weeks post-transplant, BM from primary recipients were transplanted into secondary recipients. Surprisingly, secondary recipients of blood samples collected after 2 and 5 days of G-CSF treatment had equivalent levels of donor chimerism (37.2% ± 6.6% for 2 days G-CSF and 47.1% ± 7.8% for 5 days G-CSF, n = 8 per group). Therefore, although the absolute number of RU mobilized at day 2 of G-CSF was 28-fold lower than at day 5 of G-CSF administration, more primitive serially reconstituting HSC were mobilized at equivalent levels at days 2 and 5 of G-CSF treatment. This supports our hypothesis that most potent serially reconstituting HSC are mobilized as early as day 2 of G-CSF treatment consistent with the disappearance of osteoblasts1. To test the potential of HSC remaining in the BM, BM cells from G-CSF mobilized mice were transplanted in competition with BM cells from congenic mice in steady-state. Donor chimerism at 16 weeks post-transplant showed that competitive repopulation of BM cells was severely impaired at day 5 of G-CSF treatment with the number of RU per 200,000 BM cells decreasing from 4.1 ± 1.4 in steady-state and 5.2 ± 1.6 at day 2 of G-CSF treatment, to only 0.14 ± 0.05 at day 5 of G-CSF treatment. To test whether this 29-fold decrease in competitive repopulation was due to increased HSC proliferation, we measured BrdU incorporation for the last 2.5 days prior to BM harvest as well as cell cycle analysis with Ki67 and Hoechst33342. The proportion of quiescent Lin- Sca1+ Kit+ CD48- phenotypic HSC in G0 phase decreased from 62.8 ±4.0% in steady-state to 43.5±8.2% at day 2 of G-CSF, but surged back to 80.5±1.9% and 75.1±3.5% at days 3.5 and 5 of G-CSF treatment. The proportion of HSC in G1 and S/G2/M phases followed the opposite pattern, up at day 2, down at days 3.5 and 5. This was confirmed by BrdU incorporation for 2.5 days with the number of BrdU+ cells among Lin- Sca1+ KIT+ CD48- cells rising from 35.1±4.0% in steady-state, to 51.2±4.5% at day 2 of G-CSF and going down to 18.1±1.9% at day 3.5 and 23.3±5.5% at day 5 of G-CSF. Therefore, G-CSF recruits phenotypic HSC into cell cycle within the first 2 days of administration, but HSC return to quiescence despite continuing G-CSF. Therefore decreased repopulation potential at day5 of G-CSF is not due to increased cycling. Finally, we noted that the number of Lin-Sca1+KIT+CD48-CD150+ HSC and Lin-Sca1+KIT+CD48-CD150- multipotent progenitors were reduced 2.4- and 2.8-fold respectively (p<.05) in G-CSF-mobilized BM. In conclusion, administration of G-CSF rapidly disrupts HSC niches resulting in rapid mobilization of serially-reconstituting LT-CRC as early as day 2 of G-CSF administration. Secondly, the marked reduction of competitive reconstitution potential of mobilized BM was not due to increased HSC cycling but rather to decreased number of HSC remaining in mobilized BM. Disclosures: No relevant conflicts of interest to declare.

Alternative Methods of Activation of the Stem Cell Niche Reveal a Spatial Localization of Primitive Cells in the Bone Marrow

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2396-2396
Author(s):  
Narges M Rashidi ◽  
Tassja J Spindler ◽  
Lora W Barsky ◽  
Gregor B. Adams
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Trabecular Bone ◽  
Conflicts Of Interest ◽  
Alternative Methods ◽  
Cell Populations ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Endosteal Surface ◽  
Hsc Niche

Abstract Abstract 2396 Osteoblasts are key constituents of the murine hematopoietic stem cell (HSC) endosteal niche, evidenced by the fact that increasing their activity leads to an increase in the number of HSCs. However, recent studies have also suggested a role of the bone resorbing osteoclast in the HSC niche. In these studies we wished to examine whether activation of the osteoblast or the osteoclast had differing effects on the primitive hematopoietic cell populations. To achieve this we pharmacologically activated osteoclasts and osteoblasts using receptor activator of nuclear factor-κB ligand (RANK-L) and parathyroid hormone (PTH), respectively. Our in vitro results demonstrated that both PTH and RANK-L treatment of bone marrow stromal cell populations activated osteoblasts and osteoclasts. Yet, RANK-L treatment, which is able to expand the osteoclastic population to a much higher degree, demonstrated enhanced support of primitive hematopoietic cells in cobblestone area forming cell assays. We next examined the effects of in vivo treatment with RANK-L and PTH by performing histological analyses to study the effect of these treatments on bone composition, and examining the effects on different HSC sub-populations. These data suggested that the effects of our treatment regimens were different according to the region of the bone, with RANK-L treatment having no effect or leading to a reduction in trabecular bone in the metaphysis, but an increase in bone remodeling activity at the endosteal surface of the cortical bone in the diaphysis, while PTH treatment increases bone formation and remodeling activity both in the diaphysis and the trabecular bone of the metaphysis. Correlating with this was our observations that LT-HSC frequency was increased following RANK-L and PTH treatment, yet an increased ST-HSC frequency was only observed following PTH treatment. Direct examination of HSCs in specific regions of the bone indicated that RANK-L treatment preferentially increased the LT-HSCs in the diaphysis region of the bone marrow, while PTH treatment specifically increased the ST-HSC population in the metaphysis. These data not only reveal a supportive role for osteoclasts in the HSC niche suggesting that osteoclast interaction with osteoblasts is an essential component for maintaining HSC population in the bone marrow niche, they also suggest a possible structural organization for localization of LT-HSC vs. ST-HSCs in the long bone with LT-HSCs being more localized in the endosteal surface of the diaphysis area of the long bones as oppose to the ST-HSCs having a preference for the metaphysis of these bones. Disclosures: No relevant conflicts of interest to declare.

Nfix Is Required for Hematopoietic Stem- and Progenitor Cell in Vivo Repopulating Potential.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2320-2320
Author(s):  
Per Holmfeldt ◽  
Jennifer Pardieck ◽  
Shannon McKinney-Freeman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Gene Family ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Expression Patterns ◽  
Rapid Expansion ◽  
Hematopoietic Stem ◽  
Post Transplant

Abstract Abstract 2320 Hematopoietic stem cells (HSCs) are both necessary and sufficient to sustain the complete blood system of vertebrates. Specified at several locations during fetal development, they ultimately congregate in the fetal liver for rapid expansion. Around birth HSCs relocate to the bone marrow (BM) and enter a state of cellular quiescence, cycling intermittently to supply progenitor cells, which differentiate into the distinct blood lineages. Due to their regenerative potential, HSCs are heavily utilized in the clinic for bone marrow transplants (BMTs) to treat a variety of diseases. However, a lack of suitable donors and inefficiency in recipient engraftment currently limit this life saving therapy. To improve BMT regimens, a better understanding of regulators of HSC BM engraftment is required. Recently, we examined the gene expression patterns of HSCs as they emerge throughout murine ontogeny (McKinney-Freeman et al., Cell Stem Cell, in press). We observed that the transcription factor Nfix, a member of the nuclear factor I (NFI) family of transcription factors never before linked to HSC biology, was highly expressed by both fetal liver and BM HSCs. These data suggest that Nfix may play a novel role in regulating HSC function in the BM and/or fetal liver. To test this hypothesis, HSCs were enriched from E14.5 fetal liver or adult BM (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced with lentiviruses carrying shRNAs targeting Nfix. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced BM LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfix-depleted cells. Depletion of Nfix by two independent shRNA (confirmed by Western blot analysis to deplete NFIX protein levels to <20% of baseline) resulted in a significant decrease in the repopulating activity of BM LSK cells relative to LSK cells transduced with either of two independent control shRNAs. As early as two weeks post-transplant, a 22% +/− 5% (p=0.03) reduction in repopulating activity was observed. By 16 weeks post-transplant, this reduction in repopulating potential had gradually increased to 55% +/−8% (p<0.0001) in four independent experiments. Depletion of Nfix in fetal liver-derived LSK cells resulted in a similar loss of repopulating potential. Critically, in vitro analysis of BM LSK expansion in liquid culture and differentiative potential, as analyzed by the methylcellulose based colony forming assay, revealed no differences in the activity of LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, it is unlikely that the observed decrease in BM repopulating activity is due to general cytotoxicity resulting from Nfix depletion or a block in differentiation. Concordantly, lineage analysis of peripheral blood of recipients showed no significant differences in the percentage of the major blood lineages derived from LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, the observed decrease in repopulating activity likely occurs at the level of HSCs and multipotent progenitors. In agreement with this conclusion, when BM of recipients transplanted with Nfix-depleted LSK cells are examined 4 and 16 weeks post transplant, a loss of phenotypic HSCs (LSK/CD150+/CD48-) relative to controls is evident. The loss of repopulating potential by Nfix-depleted cells as early as two weeks post-transplant suggests that Nfix may be involved in either the homing/lodgment of HSCs in the BM or their ability to expand soon after incorporation into the stem cell niche. We are presently teasing out the molecular mechanism behind this phenomenon. Furthermore, to examine a role for Nfix during HSC homeostasis, we are currently analyzing mice in which Nfix has been conditionally ablated in the hematopoietic compartment. Finally, functional analysis of two other members of the NFI gene family shown by our array analysis to be expressed by BM HSC, Nfia and Nfic, will further assess a role for this gene family in the regulation of HSCs. In summary, we have for the first time established a role for a member of the NFI gene family, Nfix, in HSC biology, as evident by a decrease in BM repopulating activity in Nfix-depleted HSCs. By dissecting the precise role of Nfix in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical BMTs. Disclosures: No relevant conflicts of interest to declare.

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.

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.

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.

Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1227-1227
Author(s):  
Elisabeth H. Javazon ◽  
Leslie S. Kean ◽  
Jennifer Perry ◽  
Jessica Butler ◽  
David R. Archer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Function ◽  
Donor Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted &gt; hemi &gt; sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

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