Abstract 020: Angiotensin Ii-induced Hypertension Has Significant Effects On Proliferation, Differentiation And Engraftment Efficacy Of Hematopoietic Stem Cell.

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Seungbum Kim ◽  
Christopher R Cogle ◽  
Michael Zingler ◽  
Edward W Scott ◽  
Mohan K Raizada
Keyword(s):  
Blood Pressure ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Inflammatory Cells ◽  
Time Lapse ◽  
Immunosuppressive Drugs ◽  
Ang Ii ◽  
Hematopoietic Stem

Cyclosporin and other immunosuppressive drugs are used in bone marrow (BM) transplantation to increase engraftment efficacy and reduce rejection. However, their chronic clinical use is closely associated with increase in blood pressure and development of hypertension (HTN). Despite these significant side effects, little is known about the influence of high blood pressure on hematopoietic stem cell (HSC) and BM activity. Thus, the objective of this study was to investigate if Ang II induced HTN exerts influence on HSC proliferation, differentiation and engraftment in the BM. Infusion of Ang II (1000ng/kg/min for 21 days) and establishment of HTN resulted in increased proliferation of HSCs as evidenced by 87% increase in Sca-1+, c-Kit+, Lin- (SKL) HSC and 254% increase in CD150+, CD48- SKL long-term HSC in the BM. Furthermore, this was associated with significant accumulation of monocytes in both BM (30% increase) and spleen (250% increase). These changes in HSC and inflammatory cells were blocked by co-infusion of Ang II and losartan (60mg/kg/day), In order to understand the effect of Ang II on HSC homing, GFP+ HSCs were injected into the lethally irradiated and saline or Ang II infused C57BL6 mice. FACS analysis of GFP+ donor derived cells showed that hypertensive animals has poor engraftment efficacy on both BM and peripheral blood (35-52% compared to saline controls). Time-lapse in vivo imaging of mouse tibia showed that HSC failed to engraft to the BM osteoblastic niche in hypertensive mice. HSCs pretreated with 100nM Ang II for 18 hours in vitro also showed significantly diminished ability (16% compared to control) to engraft in normal recipient mice. These observations demonstrate that 1) chronic Ang II induced HTN regulates HSC proliferation and impairs the homing ability and reconstitution potential of HSC in BM, 2) These effects are mediated by the AT1 receptor on HSC and 3) Ang II accelerates HSC differentiation leading the increase of inflammatory cells in BM and spleen. The results suggest that hypertensive status and BP control should be strictly taken into account in consideration for BM transplantation.

scholarly journals Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 952-962 ◽  
Author(s):  
JC van der Loo ◽  
WA Slieker ◽  
D Kieboom ◽  
RE Ploemacher
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Antigen Expression ◽  
Chimeric Mouse ◽  
Hematopoietic Stem ◽  
Colony Forming Units

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly- 6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

Abstract 156: Angiotensin Ii Regulates Hematopoietic Stem Cell Proliferation, Differentiation, and Engraftment Efficacy

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Seungbum Kim ◽  
Edward W Scott ◽  
Mohan K Raizada
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Angiotensin Ii ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Ang Ii ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Important Regulator

INTRODUCTION: Emerging evidence indicates that differentiation and mobilization of hematopoietic stem cell (HSC) are critical in the development and establishment of hypertension-linked vascular pathophysiology. This, coupled with the intimate involvement of a hyperactive renin-angiontensin system in hypertension, led us to propose the hypothesis that chronic angiotensin II (Ang II) infusion would regulate HSC proliferation and differentiation at the bone marrow level. METHODS: 1) Ang II was chronically infused into C57BL6 mice using mini-osmotic pumps (1500ng/kg/min) for 3 weeks. This resulted in an increase in MAP of 45mmHg. Bone marrow, peripheral blood and splenocytes from control and Ang II-treated mice were analyzed using FACS. 2) 0.5-3 X10 4 GFP + Sca-1 + , c-Kit + , Lin - (SKL) HSC were pre-incubated with Ang II for 24h in vitro (100μg/ml), rinsed and injected into lethally irradiated C57BL6 mice. Donor derived GFP + cells were analyzed by FACS and histology to evaluate engraftment efficiency. RESULTS: We observed a 32% decrease of HSCs in the bone marrow of Ang II treated mice. In addition, there was an 29-52% increase in the number of CX3CR1+/Gr-1- monocyte in the peripheral blood and spleen. These changes in HSC and myeloid cells were blocked by co-treatment of Losartan (60mg/kg/day, ip injection). Next, we investigated if Ang II affects HSC homing and engraftment efficacy, which are critical steps in successful bone marrow transplantation. We observed a significant delay of the homing GFP+ SKL cells that were pre-treated with Ang II in lethally irradiated recipient mice. In addition, the SKL cells treated with Ang II failed to efficiently engraft to the innate osteoblastic niche. Consistent with this observation, colony formation unit-Spleen (CFU-S) in the Ang II infused recipients was reduced to 65% compared to control mice. CONCLUSION: These observations demonstrate that hypertension induced by chronic Ang II infusion significantly impairs the engraftment ability of HSC in the bone marrow, which appears to be mediated by the AT1R on HSC and that Ang II accelerates HSC differentiation into myeloid lineage. These multifaceted roles of Ang II indicate that Ang II acts as an important regulator of HSC in the bone marrow.

Downregulation of Mpl Marks the Transition to Lymphoid-Primed Multipotent Progenitors with Gradual Loss of Granulocyte-Monocyte Potential.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2227-2227
Author(s):  
Sidinh Luc ◽  
Kristina Anderson ◽  
Ms Shabnam Kharazi ◽  
Natalija Buza-Vidas ◽  
Charlotta Boiers ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Gradual Loss ◽  
Thrombopoietin Receptor ◽  
Abrupt Changes ◽  
Lineage Priming

Abstract Evidence for a novel route of adult hematopoietic stem cell (HSC) lineage commitment through Lin−Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein residual MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Thpor), whereas LSKFlt3hiThpor− LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and co-expressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM-lymphoid progenitors with dramatically downregulated probability for committing towards MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Astrid S. Jørgensen ◽  
Viktorija Daugvilaite ◽  
Katia De Filippo ◽  
Christian Berg ◽  
Masa Mavri ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Receptor Expression ◽  
Receptor Internalization ◽  
Hematopoietic Stem ◽  
Fda Approval ◽  
Hematopoietic Stem Cell Mobilization ◽  
Cell Mobilization

AbstractFollowing the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating β-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands’ binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

New Fine Structure within the Adult Hematopoietic Stem Cell Compartment Revealed by Longterm Analysis of Mice Repopulated with Single Cells or Their In Vitro Generated Clonal Progeny.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1720-1720
Author(s):  
Brad Dykstra ◽  
David Kent ◽  
Lindsay McCaffrey ◽  
Kristin Lyons ◽  
Merete Kristiansen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Single Cells ◽  
Myeloid Cells ◽  
Hematopoietic Stem ◽  
Type 3

Abstract Assessments of hematopoietic stem cell (HSC) repopulating activity in vivo have historically relied on calculated average longterm (12–16 wk) progeny outputs using non-purified transplants, thereby precluding definitive clonal assignments of donor-derived cells. Viral marking circumvents this problem, but has not been used for large scale surveys. Heterogeneity observed in the repopulation patterns has generally been inferred to reflect stochastic processes. We now report the in vivo repopulation kinetics of 89 individual longterm repopulating cells (LTRCs) before (n=49) and after (n=40) 4 days of clonal growth in vitro. LTRCs were defined here as cells whose WBC progeny could be detected at levels of ≥1% for at least 16 wk in sublethally irradiated Ly5-congenic W41/W41 hosts. Recipients were transplanted with either freshly isolated, single lin−Rho−SP LTRCs or 4-day clones generated from similar cells in serum-free cultures (+ 300 ng/ml SF, 20 ng/ml IL-11 & 1ng/ml Flt3-L). 4, 8, 12, 16, and 24 wk post-transplant, blood samples were stained for donor-derived B, T, and myeloid cells using a procedure that identifies donor/recipient doublets and Ly6g/Mac1low cells (which have features of lymphoid rather than myeloid WBCs) to exclude false-positive myeloid events. Four distinct patterns of repopulation were revealed. Type 1 showed a delayed production of predominantly myeloid WBCs (low or undetectable before 12 wk) that increased progressively (reaching 0.4–15% of all WBCs by 16 wk). Type 2 showed a robust multilineage repopulation that remained stable or increased over time (6–84% of all WBCs at 16 wk). Type 3 also showed an initially robust pattern of multilineage repopulation (peak numbers of WBCs at 8–12 wk and 1–51% at 16 wk), but the contribution of donor-derived myeloid cells was transient (<0.5% by 16 wk). Type 4 showed a lymphoid-restricted pattern (myeloid contribution <0.5% at all time points), with repopulation levels peaking at 8 wk and decreasing thereafter (1–22% at 16 wk). Persisting granulopoiesis, indicated by a high proportion of donor-derived cells in the Ly6g/Mac1+SSChi population at 16–24 wk, clearly distinguished the type 1 and 2 patterns from types 3 and 4 which showed near or complete cessation of donor-derived granulopoiesis beyond 12 wk. Preliminary secondary transplant experiments show that donor-derived LTRCs (with and without longterm granulopoietic activity) were exclusively generated in primary recipients with type 1 and 2 repopulation patterns. Amongst the freshly isolated LTRCs, 18% (9/49) were type 1, 41% (20/49) were type 2, 22% (11/49) were type 3, and 18% (9/49) were type 4. In contrast, 4-day clones derived from cells of the same phenotype and containing LTRC activity showed a marked decrease in type 1 and type 2 activity with a corresponding increase in type 3 and type 4 activity: type 1 = 5% (2/41), type 2 = 18% (7/40), type 3 = 28% (11/40) and type 4 = 50% (20/40). Collectively, these data identify a new hierarchy of four biologically discrete states within the compartment of cells currently defined as LTRCs. Proliferation of LTRCs either in vitro or in vivo appears to induce an irreversible transition from one state to another (from Type 1 to 2 to 3 to 4), suggesting the existence of intrinsic molecular correlates for each of these states and specific mechanisms that underlie their sequential appearance.

scholarly journals Mesenchymal stromal cell remodeling of a gelatin hydrogel microenvironment defines an artificial hematopoietic stem cell niche

2018 ◽  
Author(s):  
Aidan E. Gilchrist ◽  
Sunho Lee ◽  
Yuhang Hu ◽  
Brendan A.C. Harley
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Cell Activity ◽  
Matrix Remodeling ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

AbstractHematopoietic stem cells (HSCs) reside in the bone marrow within discrete niches defined by a complex milieu of external signals including biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions. Recent studies have shown the importance of autocrine-mediated feedback of cell-secreted signals and the interplay between matrix architecture and biochemical diffusion on hematopoietic stem cell activity. Autocrine and paracrine signaling from HSCs and niche-associated mesenchymal stromal cells (MSCs) have both been suggested to support HSC maintenance in vivo and in vitro. Here we report the development of a library of methacrylamide-functionalized gelatin (GelMA) hydrogels to explore the balance between autocrine feedback and paracrine signals from co-encapsulated murine bone marrow MSCs on murine HSCs. The use of a degradable GelMA hydrogel enables the possibility for significant MSC-mediated remodeling, yielding dynamic shifts in the matrix environment surrounding HSCs. We identify a combination of an initially low-diffusivity hydrogel and a 1:1 HSPC:MSC seeding ratio as conducive to enhanced HSC population maintenance and quiescence. Further, gene expression and serial mechanical testing data suggests that MSC-mediated matrix remodeling is significant for the long-term HSC culture, reducing HSC autocrine feedback and potentially enhancing MSC-mediated signaling over 7-day culture in vitro. This work demonstrates the design of an HSC culture system that couples initial hydrogel properties, MSC co-culture, and concepts of dynamic reciprocity mediated by MSC remodeling to achieve enhanced HSC maintenance.One Sentence SummaryCoupling effects of hydrogel biotransport, heterotypic cell culture, and matrix remodeling enhances hematopoietic stem cell culture and quiescence.

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