Efficient Expansion of Human Rare Circulating Hematopoietic Stem/Progenitor Cells in Steady-State Blood Using a Polypeptide-Forming 3D Culture

2021 ◽  
Author(s):  
Yulin Xu ◽  
Xiangjun Zeng ◽  
Mingming Zhang ◽  
Xin Guo ◽  
Binsheng Wang ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cells ◽  
3D Culture ◽  
Hematopoietic Stem ◽  
Efficient Expansion

scholarly journals Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Petter Säwen ◽  
Mohamed Eldeeb ◽  
Eva Erlandsson ◽  
Trine A Kristiansen ◽  
Cecilia Laterza ◽  
...  
Keyword(s):  
Stem Cell ◽  
Steady State ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Lineage Tracing ◽  
Compensatory Mechanism ◽  
Fetal Origins ◽  
Hematopoietic Stem ◽  
Differentiation Capacity ◽  
Hsc Transplantation

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.

The Role of C5a in the Mobilization of Hematopoietic Stem/Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3472-3472
Author(s):  
Ali Jalili ◽  
Neeta Shirvaikar ◽  
Chris Korol ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Steady State ◽  
Progenitor Cells ◽  
Complement System ◽  
Cxcr4 Expression ◽  
Classical Pathway ◽  
Dependent Manner ◽  
C5a Receptor ◽  
Hematopoietic Stem ◽  
The Complement System ◽  
Deficient Mice

Abstract The complement system, a vital component of the immune system, has been shown to play a role in hematopoietic stem/progenitor cell (HSPC) trafficking. C3a is known to be important in the retention of HSPC in the bone marrow (BM) as C3a-deficient mice are good mobilizers, and C5a is important in the mobilization of HSPC because C5a-deficient mice are poor mobilizers (Stem Cells2007; 25: 3093). Further, granulocyte-colony stimulating factor (G-CSF) activates the complement system via the classical pathway, down-regulates stromal-derived factor (SDF)-1 in BM stromal cells and decreases expression of its receptor, CXCR4, in myeloid cells. In this work investigated the mechanism of C5a involvement in HSPC mobilization. Using RT-PCR and FACS we examined the expression of the C5a receptor (CD88) on mobilized peripheral blood (PB) and steady-state PB HSPC and mature white blood cells, and in vitro-expanded myeloid, erythroid, and megakaryocytic progenitor cells. We found that CD88, like the G-CSF receptor, is not expressed on BM, PB or cord blood HSPC (CD34+ cells); during CD34+ cell differentiation the expression of CD88 increases in myelocytic and megakaryocytic progenitors; and the percentage of monocytes and polymorphonuclear (PMN) cells expressing CD88 is significantly higher in mobilized than in steady-state PB. Examing the function of C5a (using flow cytometry) we found that, unlike C3a, C5a decreases CXCR4 expression in a dose-dependent manner in monocytes and PMN, but not in lymphocytes; and this effect was not seen when anti-C5a antibody was added. Interestingly, we found that G-CSF down-regulation of CXCR4 expression on PMN was partially restored by anti-C5a antibody, suggesting that the mobilizing effects of G-CSF are at least in part due to the action of C5a. Moreover, chemotaxis of PMN towards SDF-1 (chemotaxis assay) increased when these cells were stimulated with C3a but decreased with both C5a and G-CSF, reflecting the CXCR4 expression status of these cells after stimulation. We also examined the effect of C5a on matrix metalloproteinase (MMP) secretion in BM leukocytes and found that, like G-CSF, C5a increased MMP-9 and MMP-2 secretion into media (zymography). Since the SDF-1/CXCR4 axis plays an integral role in the retention of HSPC in the BM, we conclude that C5a promotes mobilization by disrupting this axis, as well as increasing MMP-9 and MMP-2 secretion by BM leukocytes, thereby allowing egress of HSPC into the PB.

scholarly journals Sphingosine-1-phosphate-Mediated Mobilization of Hematopoietic Stem/Progenitor Cells during Intravascular Hemolysis Requires Attenuation of SDF-1-CXCR4 Retention Signaling in Bone Marrow

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Kasia Mierzejewska ◽  
Yuri M. Klyachkin ◽  
Janina Ratajczak ◽  
Ahmed Abdel-Latif ◽  
Magda Kucia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Blocking Agent ◽  
Intravascular Hemolysis ◽  
Hematopoietic Stem ◽  
Sphingosine 1 Phosphate ◽  
Retention Signal ◽  
Synergistic Increase

Sphingosine-1-phosphate (S1P) is a crucial chemotactic factor in peripheral blood (PB) involved in the mobilization process and egress of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM). Since S1P is present at high levels in erythrocytes, one might assume that, by increasing the plasma S1P level, the hemolysis of red blood cells would induce mobilization of HSPCs. To test this assumption, we induced hemolysis in mice by employing phenylhydrazine (PHZ). We observed that doubling the S1P level in PB from damaged erythrocytes induced only a marginally increased level of mobilization. However, if mice were exposed to PHZ together with the CXCR4 blocking agent, AMD3100, a robust synergistic increase in the number of mobilized HSPCs occurred. We conclude that hemolysis, even if it significantly elevates the S1P level in PB, also requires attenuation of the CXCR4-SDF-1 axis-mediated retention in BM niches for HSPC mobilization to occur. Our data also further confirm that S1P is a major chemottractant present in plasma and chemoattracts HSPCs into PB under steady-state conditions. However, to egress from BM, HSPCs first have to be released from BM niches by blocking the SDF-1-CXCR4 retention signal.

Cell-Intrinsic and Cell-Extrinsic Involvement Of C/EBPβ In The Regulation Of Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1202-1202
Author(s):  
Akihiro Tamura ◽  
Hideyo Hirai ◽  
Yoshihiro Hayashi ◽  
Asumi Yokota ◽  
Atsushi Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Leucine Zipper ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
The Difference

Abstract Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein β (C/EBPβ), a leucine zipper transcription factor, in emergency granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPβ is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem/progenitor cells (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPβ is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and leukemic stem cells exhaustion in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPβ plays important roles in normal hematopoietic stem cells (HSCs). Here we investigated the cell-intrinsic and -extrinsic function of C/EBPβ in the regulation of HSCs by analyzing C/EBPβ knockout (KO) mice. At steady state, no obvious defects have been reported in hematopoiesis of C/EBPβ KO mice. Accordingly, the frequencies of long-term and short-term HSCs and various kinds of progenitor cells in bone marrows (BM) of C/EBPβ KO mice were identical to those in BM of wild type (WT) mice. To examine the functional consequences of C/EBPβ deletion, competitive repopulation assay was performed. In brief, 5x105 BM cells from WT or C/EBPβ KO mice (CD45.2+) and the same number of competitor CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the chimerisms of CD45.2+ cells in the peripheral blood of the recipient mice were monitored monthly. The chimerisms of C/EBPβ KO cells were significantly lower than that of WT cell at 1 month after transplantation and the differences were maintained thereafter (Figure A). In order to elucidate the reason for the difference, homing ability of C/EBPβ KO cells were assessed. Lineage depleted CD45.2+ WT or C/EBPβ KO BM cells together with the equal number of lineage negative CD45.1+ BM cells were transplanted into lethally irradiated CD45.1+ mice and the frequencies of CD45.2+ cells were analyzed 16 hours after transplantation. The frequencies of CD45.2+ WT and C/EBPβ KO donor cells in the recipient BMs were identical and the data indicated that the differences in the chimerisms after primary BM transplantation were due to the difference in the initial expansion of transplanted cells after equivalent levels of homing. To see the roles of C/EBPβ in hematopoiesis under stressed conditions, CD45.1+ mice were transplanted with CD45.2+ WT or C/EBPβ KO BM cells with equal numbers of CD45.1+ BM cells and these mice were administered with 150mg/kg 5-fluorouracil (5-FU) once a month and the chimerisms of peripheral blood were monitored every time before the next 5-FU administration. In consistent with the results mentioned above, the frequencies of CD45.2+ C/EBPβ KO cells were significantly lower than those of CD45.2+ WT cells 1 month after transplantation. After repetitive administration of 5-FU, however, the chimerisms of CD45.2+ C/EBPβ KO cells gradually caught up with those of CD45.2+ WT cells, suggesting that C/EBPβ is involved in the exhaustion of HSCs under stressed conditions (Figure B). To explore the functions of C/EBPβ in hematopoietic microenvironments, 1x106 CD45.1+ BM cells from WT mice were transplanted into irradiated (5Gy or 7Gy) WT or C/EBPβ KO mice (CD45.2+). All the WT recipient mice survived after 5Gy or 7Gy irradiation (4/4 and 4/4, respectively). In contrast, only 2/4 and 1/4 C/EBPβ KO recipient mice survived after 5Gy or 7Gy irradiation, respectively. We are currently trying to identify the cells expressing C/EBPβ in BM microenvironments and investigating the mechanisms for the higher sensitivity of C/EBPβ KO mice to irradiation. In summary, these data suggested that C/EBPβ is required for initial expansion of hematopoietic stem/progenitor cells at the expense of HSCs under stressed conditions, while it is dispensable for maintenance of HSCs at steady state. We are now investigating the cellular and molecular targets of C/EBPβ in HSC regulation and would like to elucidate the cell-intrinsic and cell-extrinsic mechanisms in regulation of the homeostasis of hematopoietic system by C/EBPβ. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Derived Mesenchymal Cells Secrete Granulopoietic Cytokines upon Danger Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4115-4115
Author(s):  
Stefan Wirths ◽  
Stefanie Bugl ◽  
Markus P. Radsak ◽  
Melanie Märklin ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Feedback Loop ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Caspase 1 ◽  
Stem And Progenitor Cells ◽  
Myd88 Signaling

Abstract Granulopoietic homeostasis is regulated at steady-state to supply sufficient numbers of pooled and circulating neutrophils to maintain barrier function against commensal flora. In addition, upon pathogenic microbial challenge, an increased formation of neutrophils is induced, termed ‘emergency granulopoiesis’. Antibody-mediated reduction of neutrophil numbers in steady-state induces a feedback loop leading to an increase of bone marrow granulopoiesis with expansion of hematopoetic stem and progenitor cells. This feedback loop was demonstrated to depend on TLR4 and TRIF, but not MyD88 signaling (Bugl et al. Blood 2013). In contrast, emergency granulopoiesis was shown to be dependent on MyD88 signaling in endothelial cells (Boettcher et al. Blood 2014). Bone marrow mesenchymal stromal cells (MSC) are niche-forming cells, harboring and regulating hematopoiesis. Upon steady-state neutropenia an increase of niche size was observed. Here we investigated, whether niche-forming MSC act as sensors of pathogen-associated molecular patterns (PAMPs) and induce granulopoietic cytokines to stimulate expansion of adjacent hematopoietic stem and progenitor cells. MSC of C57BL/6 and TLR4-KO mice were cultured in vitro and treated with LPS for 24 hours. Cells were harvested and qRT-PCR for G-CSF, TLR4, MyD88, TRIF, GM-CSF, IL-1β, IL-18 and Casp-1 was performed After treatment with LPS, RNA of granulopoietic cytokines G-CSF and GM-CSF were massively up regulated in MSC of WT mice. Upstream regulating, inflammasome components IL-1ß and caspase-1 RNA levels increased as well, with little changes in IL-18, TLR4, MyD88 and TRIF. Unexpectedly, TLR4-KO MSC up regulated transcription of IL-1β and G-CSF upon LPS stimulation as well, and caspase-1 was found to be strongly up-regulated in unstimulated TLR4-KO compared to WT MSC. In summary, bone marrow stromal cells are found to be PAMP-sensing and secrete cytokines that regulate granulopoiesis. TLR4-independent sensing of LPS by MSC might correspond to the alternative noncanonical inflammasome pathway recently described (Kayagaki et al. Science 2013). Disclosures No relevant conflicts of interest to declare.

C/EBPβ Promotes Initial Expansion and Exhaustion of Hematopoietic Stem and Progenitor Cells in Response to Hematopoietic Stresses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5126-5126
Author(s):  
Atsushi Sato ◽  
Hideyo Hirai ◽  
Akihiro Tamura ◽  
Asumi Yokota ◽  
Yoshihiro Hayashi ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cells ◽  
Leucine Zipper ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Chronic Phase ◽  
Early Time ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions. At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state. To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p < 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation. In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation. Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress. From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals HSCs Contribute Actively to Native Multilineage Hematopoiesis but With Reduced Differentiation Capacity Upon Aging

2018 ◽  
Author(s):  
Petter Säwén ◽  
Mohamed Eldeeb ◽  
Eva Erlandsson ◽  
Trine A Kristiansen ◽  
Cecilia Laterza ◽  
...  
Keyword(s):  
Stem Cells ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Lineage Tracing ◽  
Compensatory Mechanism ◽  
Hematopoietic Stem ◽  
Differentiation Capacity

ABSTRACTA hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. It is well established that adult hematopoietic stem cells (HSCs) are active contributors to these processes after transplantation, yet their role in native hematopoiesis has recently been called into question. Here, we use inducible lineage tracing from genetically marked adult HSCs to explore their roles in the steady state. We show that adult HSCs contribute robustly to all lineages via intermediate progenitor cells, but with neglible production of hematopoietic cells with a known fetal origin. We further reveal that the timing for regeneration of distinct blood lineages varies substantially. Finally, HSC contribution to multilineage hematopoiesis in aged animals declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for a reduced differentiation capacity with age.

Maintenance and Expansion of Hematopoietic Stem/Progenitor Cells in a Biomimetic Hematopoietic Cells Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4115-4115
Author(s):  
Ting Liu ◽  
Jing Tan ◽  
Li Hou ◽  
Wentong Meng ◽  
Yuchun Wang
Keyword(s):  
Progenitor Cells ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
3D Culture System ◽  
2D System

Abstract As main hematopoietic organ, bone marrow have three dimensional microenvironment for hematopoietic stem/progenitor cells grow in, so call “hematopoietic cells niche”, which are composed by stromal cells and extracelluar matrix. The interactions of cell to cell and cell to matrix between stem/progenitor cells with hematopoietic niche are facilitated by its three dimensional conformation. The biology behaviors of hematopoietic stem cells are mediate by many signal transductions between stem/progenitor cells with their corresponding microenvironment. Now there are strong evidence from animal model study suggests that osteoblasts might play an essential role in creation of a hematopoietic stem cell niche and thereby regulation of stem cell maintenance, proliferation, and maturation. In light of the structure-function relationship of bone marrow topography, we conceived a biomimetic culture system (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts to simulate the effects of hematopoietic osteobalst niche. CD 34+ cells or mononuclear cells separated from umbilical cord blood were cultured for 2∼5 weeks in the 3D culture system and also in a conventional 2D culture system as control without additional cytokine supplement. Based on our results, higher expression of extracelluar matrix and N-cadherin were observed in 3D culture system compared to 2D system. At 2 weeks culture, 3D culture system showed higher number of CD34+ cells and CD34+/CD38- cells when compared with the input (P<0.05), the increased cells were predominant CD34+/CD38-cells. Although CD34+ cells were decreased at 5 weeks culture; nevertheless, CD34+/CD38- cells were still maintained at high level. We also observed that imbedding MNCs with a higher percentage of CD34+/CD38-cells cultured in 3D system (P<0.05), which may represent a down regulation of CD38 phenotype during culture. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced higher expansion of CFU progenitors than 2D system (7.13–8.89 times vs. 1.22–1.31times) after 2 weeks culture. Of note, the colony distribution of 3D system manifested higher percentage of BFU-E and CFU-GEMM, while 2D system showed mainly CFU-GM. LTC-IC represents the primitive progenitor, 3D system showed a 6.2 times increment over input after 2 weeks culture. Furthermore, it was competent to maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. This study demonstrated that our 3D culture system constructed with the bio-derived bone composited with induced osteoblast is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. It may open a new avenue to study HSCs/HPCs behaviors and to achieve sustained primitive progenitor cell expansion.

The Combination of AMD3100 + G-CSF Restores the Ineffective Hematopoietic Stem Cell Mobilization with G-CSF Alone in a Thalassemic Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3234-3234
Author(s):  
Evangelia Yannaki ◽  
Nikoleta Psatha ◽  
Maria Demertzi ◽  
Evangelia Athanasiou ◽  
Eleni Sgouramali ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Mouse Model ◽  
Progenitor Cells ◽  
State Condition ◽  
Hematopoietic Stem ◽  
Steady State Condition ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 3234 Poster Board III-171 Gene therapy has been recently postulated as a realistic therapeutic potential for thalassemia and the mobilized autologous hematopoietic stem cells (HSCs) may represent the preferable source of stem cells for genetic modification due to the higher yield of HSCs compared to conventional bone marrow (bm) harvest. We have previously shown (manuscript under revision) that G-CSF mobilization in the HBBth-3 thalassemic mouse model is less efficient compared to normal C57Bl6 strain, mainly due to increased trapping of hematopoietic stem (Lin-sca-1+ckit+–LSK) and progenitor cells (CFU-GM) in the enlarged thalassemic spleen. The novel mobilizer, AMD3100 (plerixafor, mozobil), has been shown to reversibly bind to CXCR4 and inhibit the interaction between SDF-1 and CXCR4 within the bm microenvironment, resulting in the egress of CD34+ cells into the circulation of healthy donors and cancer patients. The addition of AMD to G-CSF results in even greater increases in circulating CD34+cells. We explored in the current study whether AMD alone or in combination with G-CSF improves the mobilization efficiency of thalassemic mice. C57 and HBBth-3 mice received G-CSF-alone at 250microgr/kgX7 days, AMD-alone at 5mg/kgX3 days or the combination of two with AMD administered in the evening of days 5-7 of G-CSF administration. Hematopoietic tissues (blood, bm, spleen) were collected and the absolute LSK and CFU-GM numbers were calculated based on their frequency within tissues (by FCM and clonogenic assays) in relation to the individual cell count per tissue. AMD-alone didn't significantly affect the HSC yield as compared to G-CSF mobilization in thal mice (LSK/μl blood: 103±85 vs 69±26 p=ns), although it significantly increased the circulating Colony Forming Cells (CFU-GM/ml blood: 1205±533 vs 330±87, p=0,05). In contrast, the AMD+G-CSF combination significantly improved the mobilization efficiency of HBBth-3 mice over the G-CSF-treated group (LSK cells/μl blood: 224±104 vs 69±26 p=0,04, CFU-GM/ml blood: 1671±984 vs 330±87 p=0,05, respectively) at levels comparable to normal mice treated with G-CSF (LSK cells/ μl blood: 241±167, CFU-GM/ml blood: 1235±1140, respectively). AMD induced a “detachment” of stem cells from the bm because reduced numbers of bm LSK cells were counted in the AMD-alone group as compared to the untreated group (LSK/2 femurs×103: 692±429 vs 1687±1016, respectively, p=0,05). This was in contrast to the marrow hyperplasia caused by G-CSF over the steady-state condition (LSK/2 femurs×103: 2684±1743 vs 1687±1016 p=0,02 / CFU-GM/2femurs:111.841±15.391 vs 76.774±31.728 p=0,01). Consequently, the combination of AMD+G-CSF resulted in increased numbers of circulating stem and progenitor cells without inducing marrow hyperplasia as compared to steady-state condition (LSK/2femurs×103: 1681±862 vs 1686±1017, p=ns / CFU-GM/2femurs: 76.774±31.728 vs 82.905±26.277, p=ns). AMD, also in contrast to G-CSF, did not cause increased trapping of stem and progenitor cells in the spleen compared to the untreated condition (LSK cells/spleen×103: 4738±2970 vs 8303±4166 p=ns / CFU-GM/spleen:146.269±93.174 vs 98.518±25.549, p=ns). However, the combination of AMD+G-CSF still resulted in splenic sequestration of progenitor cells (CFU-GM/spleen: 412.176±157.417 vs 98.518±25.549, p=0,0003) but not of LSK cells (LSK cells/spleen×103: 10.200±7.260 vs 8.300±4.166 p=ns). Overall, the combination of AMD3100+G-CSF seems to restore the less efficient mobilization in a thalassemic mouse model. This combination may prove beneficial in a GT setting for obtaining the high numbers of HSCs needed for genetic correction. In addition, the combination of AMD3100+G-CSF, by avoiding the marrow hyperplasia induced by G-CSF alone, indicates a better safety profile because it will not further burden the hyperplastic –due to the increased erythroid demand and the intramarrow destruction of erythroblasts-thalassemic bone marrow. Disclosures No relevant conflicts of interest to declare.

Dendritic Cell Homeostasis Is Maintained by Non-Hematopoietic and T Cell-Produced Flt3-Ligand In Steady-State and During Immune Responses.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1547-1547
Author(s):  
Chandra Sekhar Boddupalli ◽  
Dior Baumjohann ◽  
Tim Sparwasser ◽  
Markus G Manz
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Progenitor Cells ◽  
T And B Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1547 Lymphoid tissue dendritic cells (DCs) have a short life-span of a few days and need to be continuously replenished from hematopoietic stem and progenitor cells. Flt3-Ligand (Flt3L) plays non-redundant role in development of DCs (McKenna. H.J. et al., Blood; 2000). Previously we found that Flk2 (fetal liver kinase-2), the cognate receptor for Flt3L is expressed on early dendritic cell progenitors and Flt3L-Flk2 signalling efficiently supports DC development from early progenitors to steady-state DCs in mice and men (Karsunky, H. et al., J Exp Med; 2003; Chicha L. et al. J Exp Med; 2004). Flk2 is also expressed on mature steady-state lymphoid organ DCs; however its function on mature cells remains to be determined. Flt3L is expressed in almost all the tissues in both mice and men (Hannum, C. et al., Nature; 1994) and this cytokine is critical in the maintenance of DC/T regulatory (Treg) cell homeostasis (Darrase-Jéze. G et al., J Exp Med; 2009; Swee LK et al., Blood; 2009; Manz MG, Blood 2009). However, the precise cellular source of Flt3L and the regulation of production in steady-state and immune responses in vivo is not well understood. Genetic ablation of the Flk2 receptor lead to 10-fold elevated Flt3L levels in the serum of mice. To evaluate if hematopoietic or non-hematopoietic cells are the main consumers of Flt3L in vivo, we generated bone marrow chimeras by transplanting wild type (WT) or Flt3L-/- c-Kit+ hematopoietic stem and progenitor cells into lethally irradiated Flk2-/- mice. This demonstrated that hematopietic progenitors and DCs expressing Flk2 receptor are the main consumers of Flt3L in vivo. Previously we showed that in vivo Flk2 tyrosine kinase inhibition and consecutive DC reduction lead to 10fold elevated levels of serum Flt3L (Tussiwand. R. et al., J Immunol; 2005). By using CD11c DTR mice (Zaft, T. et al., J Immunol; 2005) in which diphtheria toxin (DT) receptor is cloned under the CD11c promoter and treatment of mice with DT lead to selective depletion of DCs we here show that ablation Flk2 expressing DCs lead to immediate, about 4-fold elevated serum Flt3L levels in mice. However, we observed no change in mRNA expression of Flt3L, which strongly indicates that Flk2 expressed on DCs is acting as “scavenger” for Flt3L. We then studied sources of Flt3L in vivo. To this end we generated bone marrow chimeras by transplanting WT c-Kit+ hematopoietic stem and progenitor cells in to lethally irradiated Flt3L-/- hosts and vice versa (WT to Fllt3L-/-, Flt3L-/- to WT), and found that in vivo DC homeostasis can be achieved by non-hematopoietic and to lesser extend by hematopoietic cell produced Flt3L. Furhtermore, we found that compared to other hematopoietic cells Flt3L mRNA is highly expressed in lymphocytes (T and B cells) and in lymphoid tissues like thymus, spleen and lymph nodes. We thus used bone marrow c-Kit+ hematopoietic stem and progenitor cells from mice that lack T and B cells (Rag1-/-) or that lack T cells (CD3ε-/-) as donors to transplant lethally conditioned Flt3L-/- mice, and found that Flt3L produced by T and B cells is necessary to support DC development in non hematopoietic Flt3L deficient mice. Using BrdU incorporation we evaluated the functional relevance of Flt3L produced by T cells in an ongoing immune response. Experiments revealed that in lymph nodes with proliferating T cells producing Flt3L a higher percent of BrdU+ DCs, i.e. DCs derived from proliferating progenitors were detected. This indicates that Flt3L produced by T cells in an ongoing immune response helps in faster regeneration of DCs from DC committed progenitors. Earlier it has been shown that Treg ablation in Foxp3-DTR mice lead to expansion of DCs in lymph nodes and spleen through Flk2 mediated pathway (Liu, K. et al., Science; 2009); however, the source of Flt3L remained unknown. Here we provide evidence that Treg ablation leads to activation and proliferation of CD4+ T cells that in turn release Flt3L to enhance DC development. These key observations provide insight into the regulation of DC homeostasis and function via tailored adaptation of the Flt3L cytokine milieu by non-hematopoietic and T cells during steady state and during adaptive immune responses. Supported by the Swiss National Science Foundation (310000-116637) and the European Commission FP6 Network of Excellence initiative (LSHB-CT-2004-512074 DC-THERA) Disclosures: No relevant conflicts of interest to declare.

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