scholarly journals Effect of Mesenchymal Stem Cell-derived Microvesicles on Megakaryocytic Differentiation of CD34+ Hematopoietic Stem Cells

2020 ◽  
Vol 10 (2) ◽  
pp. 315-322
Author(s):  
Sara Aqmasheh ◽  
Karim Shamsasenjan ◽  
Elham Khalaf Adeli ◽  
Aliakbar Movassaghpourakbari ◽  
Parvin Akbarzadehlaleh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Significant Expression ◽  
Late Stages ◽  
Megakaryocyte Differentiation

Purpose: Mesenchymal stem cells (MSCs) release hematopoietic cytokines, growth factors, and Microvesicles (MVs) supporting the hematopoietic stem cells (HSCs). MVs released from various cells, playing a crucial role in biological functions of their parental cells. MSC-derived MVs contain microRNAs and proteins with key roles in the regulation of hematopoiesis. Umbilical cord blood (UCB) is a source for transplantation but the long-term recovery of platelets is a main problem. Therefore, we intend to show that MSC-MVs are able to improve the differentiation of UCB-derived CD34+ cells to megakaryocyte lineage. Methods: In this descriptive study, MSCs were cultured in DMEM to collect the culture supernatant, which was ultracentrifuged for the isolation of MVs. HSCs were isolated from UCB using MACS method and cultured in IMDM supplemented with cytokines and MVs in three different conditions. Megakaryocyte differentiation was evaluated through the expression of specific markers and genes after 72 hours, and the data was analyzed by t test (P<0.05). Results: The expression of specific megakaryocyte markers (CD41 and CD61) in the presence of different concentrations of MSC-MVs did not show any significant difference. Also, the expression of specific genes of megakaryocyte lineage was compared with control group. The expression of GATA2 and c-Mpl was significantly increased, GATA1 was not significantly decreased, and FLI1 was significantly decreased. Conclusion: MSC-MVs could improve the expression of specific megakaryocyte genes; however, there was no significant expression of CD markers. Further studies, including the evaluation of late stages of megakaryocyte differentiation, are required to evaluate platelet production and shedding

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals Separation of hematopoietic stem cells into two populations and their characterization

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 91-95 ◽  
Author(s):  
H Ogata ◽  
S Taniguchi ◽  
M Inaba ◽  
M Sugawara ◽  
Y Ohta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Reconstruction Efficiency ◽  
G0 Phase ◽  
Long Term Observation ◽  
Two Populations

Two populations of hematopoietic stem cells (HSC) in mouse bone marrow (BM) are defined on the basis of the presence or absence of interleukin- 3 (IL-3) receptor-associated antigen (IL-3RAA). HSC were purified by depletion of mature lymphoid-lineage cells followed by collection of the low-density fraction and sorting of wheat germ agglutinin-binding (WGA+) cells using a fluorescein-activated cell sorter. WGA+ cells were further separated into two populations (IL-3RAA+/WGA+ and IL-3RAA- /WGA+) by a monoclonal antibody (MoAb) against IL-3RAA. IL-3RAA+/WGA+ cells formed CFU-S on day 8; this population consisted mainly of cells in the cycling phase. IL-3RAA-/WGA+ cells form CFU-S on day 12; this population consisted mainly of dormant cells (cells in the G0 phase). When two populations obtained from C3H/HeN mice were injected into lethally irradiated (C57BL/6 x C3H/HeN)F1 mice, donor-derived cells in the peripheral blood (PB) appeared significantly earlier in mice injected with IL-3RAA+/WGA+ cells than in those injected with IL-3RAA- /WGA+ cells, whereas the reconstruction efficiency of IL-3RAA-/WGA+ cells had overtaken that of IL-3RAA+/WGA+ cells 6 weeks after injection. Long-term observation showed no significant difference between these two populations, however. The radioprotective ability (RPA) (30-day survival) of these two populations was therefore compared. The RPA of IL-3RAA-/WGA+ cells was significantly higher than that of IL-3RAA+/WGA+ cells. These findings therefore suggest that the former population is more primitive.

Improved Engraftment of Human Hematopoietic Stem Cells in NOD/SCID Mice by the Antioxidant, N-Acetyl-L-Cysteine

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1884-1884
Author(s):  
Linping Hu ◽  
Yingdai Gao ◽  
Yanfeng Liu ◽  
Hui Cheng ◽  
Jing Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Scid Mice ◽  
Mouse Strains ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Treated Drinking Water

Abstract Abstract 1884 Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice are widely used as a human-mouse xeno-transplant model for assessing engraftment of human hematopoietic stem cells (HSCs). Optimizing this model and understanding the variables that affect engraftment are critical to correctly interpret the engraftment results of human HSCs in NOD/SCID recipients. However, the engraftment efficiency of human HSCs remains very low in NOD/SCID mice. Larger efforts have been devoted to improving engraftment by increasing the immune-deficiency of the mice, but less effort has been made toward other negative parameters in the host. Our preliminary study showed that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in the bone marrow (BM) in comparison with other commonly used mouse strains (C57BL/6 and BALB/C). Given the previous studies by us and others showing that excessive ROS could impair the function of HSCs and that antioxidants were able to overcome the exhaustion of mouse HSCs in transplant recipients, we hypothesized that the poor engraftment of human hematopoietic cells in NOD/SCID recipients may be partially attributed to higher levels of ROS in NOD/SCID BM and a reduction of ROS by antioxidants may improve the engraftment of human HSCs in NOD/SCID mice. To test this hypothesis, NOD/SCID (8- to 12-week-old) mice were injected subcutaneously daily with an antioxidant, N-acetyl-L-cysteine (NAC) or PBS (control) for two weeks before being irradiated with 200 cGy from a cesium-137 source at 70cGy/min. Different doses of CD34+ cells or highly-enriched HSCs from human cord blood (CB) were injected through the tail vein or into the right tibia of the mice. The mice were maintained on NAC treated drinking water following injection, then sacrificed 12–14 weeks after transplantation to measure the engraftment levels of different hematopoietic cell lineages. We found that treatment with NAC was able to lower the levels of ROS in NOD/SCID BM. At the highest dose of injected CD34+ cells (>5×105), the NAC treated recipients displayed a significant increase of engraftment (2.1-fold) when compared with the control group (Control vs. NAC treated recipients: 11.04±3.11% vs. 23.21±4.0%, p=0.0224; n=20/each). This improvement was even more significant when injected cell numbers were reduced (2×105 and 1×105 had 3.9- and 4.9- fold higher engraftment, respectively, in NAC treated recipients), thus suggesting that saturating levels of HSCs may ease the anti-oxidant effect on engraftment. Furthermore, we also demonstrated higher levels of overall engraftment and multi-lineage differentiation of human HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+Rholow) with a limiting dilution analysis. In comparison with the control mice, NAC treated recipients displayed 2.5-, 3.5-, and 5.7-fold increases in engraftment in the injected tibia (IT), BM and spleen, respectively. The frequency of SCID-repopulating cell (SRC) in IT was approximately 3.0-fold higher in NAC treated mice than in control mice (1 in 108 vs. 1 in 36). Similar improvements (4.1- to 7.9- fold) in SRC frequencies were also detected in BM and spleen. Notably, NAC increased the probability of positive engraftment when a single human HSC was directly transplanted into the BM of NOD/SCID mice. In summary, our current study uncovers a previously unappreciated negative effect of ROS in the human-NOD/SCID xenotransplant model and reduction of ROS via antioxidants such as NAC may significantly enhance the engraftment of human hematopoietic stem cells in NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Separation of hematopoietic stem cells into two populations and their characterization

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 91-95 ◽  
Author(s):  
H Ogata ◽  
S Taniguchi ◽  
M Inaba ◽  
M Sugawara ◽  
Y Ohta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Reconstruction Efficiency ◽  
G0 Phase ◽  
Long Term Observation ◽  
Two Populations

Abstract Two populations of hematopoietic stem cells (HSC) in mouse bone marrow (BM) are defined on the basis of the presence or absence of interleukin- 3 (IL-3) receptor-associated antigen (IL-3RAA). HSC were purified by depletion of mature lymphoid-lineage cells followed by collection of the low-density fraction and sorting of wheat germ agglutinin-binding (WGA+) cells using a fluorescein-activated cell sorter. WGA+ cells were further separated into two populations (IL-3RAA+/WGA+ and IL-3RAA- /WGA+) by a monoclonal antibody (MoAb) against IL-3RAA. IL-3RAA+/WGA+ cells formed CFU-S on day 8; this population consisted mainly of cells in the cycling phase. IL-3RAA-/WGA+ cells form CFU-S on day 12; this population consisted mainly of dormant cells (cells in the G0 phase). When two populations obtained from C3H/HeN mice were injected into lethally irradiated (C57BL/6 x C3H/HeN)F1 mice, donor-derived cells in the peripheral blood (PB) appeared significantly earlier in mice injected with IL-3RAA+/WGA+ cells than in those injected with IL-3RAA- /WGA+ cells, whereas the reconstruction efficiency of IL-3RAA-/WGA+ cells had overtaken that of IL-3RAA+/WGA+ cells 6 weeks after injection. Long-term observation showed no significant difference between these two populations, however. The radioprotective ability (RPA) (30-day survival) of these two populations was therefore compared. The RPA of IL-3RAA-/WGA+ cells was significantly higher than that of IL-3RAA+/WGA+ cells. These findings therefore suggest that the former population is more primitive.

Robo4/Magic Roundabout Is a Novel Surface Marker for Murine and Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 682-682
Author(s):  
Fumi Shibata ◽  
Yuko Goto-Koshino ◽  
Miyuki Ito ◽  
Yumi Fukuchi ◽  
Yoshihiro Morikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cd34 Cells ◽  
Human Hscs

Abstract A variety of cell surface markers such as c-Kit, Sca-1, CD34 and Flt-3 have been utilized to prospectively isolate murine or human hematopoietic stem cells (HSCs). While murine HSCs were shown to be highly enriched in CD34−c-Kit+Sca-1+Lineage- (CD34−KSL) fraction, this population is still not homogeneous for long-term HSCs. In human, CD34+ cells are regarded as crude HSC fraction and used for clinical applications. However, quiescent human HSCs are also found in CD34− fraction, indicating that CD34 is not a bona fide marker for human HSC. Thus, novel surface markers that can be used to purify human or murine HSCs to homogeneity need to be identified. Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are predominantly expressed in nervous system. Slit2, a ligand for Robo, is a large leucine-rich repeat-containing secreted protein that is also expressed in brain. By binding with Robo, Slit2 acts as a repellant for axon guidance of developing neurons and they are critical for correct wiring of neuronal network. Robo family comprises four family members, Robo1 – Robo4, and Robo4 is distinct in that it is expressed specifically in endothelial cells, but not in brain. In this study, we investigated Robo4 for its possible application for HSC identification in murine and human hematopoietic system. By RT-PCR, Robo4 was specifically expressed in murine KSL fraction, and was not expressed in lineage positive cells and various progenitors such as common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP) and common lymphoid progenitor (CLP). Moreover, the expression of Robo4 was highest in side population of KSL cells (KSL-SP), and moderate in KSL-main population (KSL-MP) cells. Monoclonal antibody raised against Robo4 identified its high expression in KSL cells by FACS. FACS analysis of human cord blood cells revealed that Robo4 is highly expressed in CD34+ cells, and CD34+Robo4high population fell into CD38− fraction, which enriches human HSCs. Bone marrow transplantation experiments revealed that Robo4+ fraction of murine KSL cells had long-term repopulating activity, while Robo4−KSL cells not. Although both Robo4+ and Robo4− CD34−KSL cells repopulated murine hematopoietic system for long-term, Robo4+CD34−KSL cells achieved higher chimerism after repopulation compared with Robo4−CD34−KSL. To investigate the physiological role of Robo4 in HSC homeostasis, we next examined the expression of Slit2 in hematopoietic system. Interestingly, Slit2 is specifically expressed in bone marrow stromal cells, but not in hematopoietic cells. Moreover, Slit2 is induced in osteoblasts, a critical cellular component composing HSC niche, in response to myelosuppressive stress such as 5FU treatment. These results indicate that Robo4 is expressed in murine and human hematopoietic HSCs and useful for HSC purification, and Robo4 - Slit2 system may play a role in HSC physiology in niche environment under hematopoietic stress.

Novel lentiviral vectors displaying “early-acting cytokines” selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3386-3395 ◽  
Author(s):  
Els Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Surface Display ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells, that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying “early-acting cytokines” on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34+ cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34+ cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34+ cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.

scholarly journals Etoposide-Initiated MLL Rearrangements Detected at High Frequency in Human Primitive Hematopoietic Stem Cells with In Vitro and In Vivo Long-Term Repopulating Potential.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 98-98 ◽  
Author(s):  
Jolanta Libura ◽  
Marueen Ward ◽  
Grzegorz Przybylski ◽  
Christine Richardson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
P Value ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Rearrangements involving the MLL gene locus at chromosome band 11q23 are observed in therapy-related acute myeloid leukemia and myelodysplastic syndromes following treatment with topoisomerase II (topoII) inhibitors including etoposide. We have shown that one hour of etoposide exposure (20–50 μM) stimulates stable MLL rearrangements in primary human CD34+ cells and that the spectrum of repair products within MLL gene is broader than so far described (Libura et al, Blood, 2005). Clinical data suggest that MLL-associated malignant leukemias originate within primitive hematopietic stem cells capable of differentiation into all hematopoietic lineages and repopulation of myelo-ablated hosts. These cells can be analyzed using the in vivo NOD-SCID mouse model as well as the in vitro long-term culture initiating cell (LTC-IC) assay. We adopted our in vitro CD34+ cell culture model to investigate the impact of etoposide exposure on the most primitive hematopoietic stem cells using parallel assays for LTC-IC and NOD-SCID Repopulating Cells (SRC). Following etoposide exposure (20–50 μM for 1 hour), and 48–96 hours recovery in vitro, untreated control and etoposide-treated CD34+ cells were either seeded in LTC-IC with a supportive feeder layer (Stem Cell Technologies, Inc.), or injected into NOD-SCID mice (0.1–1.5x106 cells per mouse). After 12 weeks, both LTC-IC cultures and bone marrow cells from NOD-SCID mice were seeded in methylcellulose media supplemented with growth factors that promote only human cell colony formation. An increased number of colonies in etoposide-treated samples was obtained from LTC-IC cultures in 3 out of 5 experiments (p value&lt;0.05). This increase in colony number was more dramatic in etoposide-treated samples from NOD-SCID bone marrow (57 versus 0, 8 versus 0). These data demonstrate that etoposide exposure can significantly alter the potential of early hematopoietic stem cells to survive and proliferate both in vitro and in vivo. Injection of as few as 3x105 CD34+ cells into a NOD-SCID mouse was sufficient to obtain methylcellulose colonies, suggesting that this method can be used for the analysis of cells obtained from a single patient sample. Mutation analysis of human methylcellulose colonies derived from both LTC-IC and NOD-SCID was performed by inverse PCR and ligation-mediated PCR followed by sequencing. This analysis revealed that rearrangements originating within the MLL breakpoint cluster region (bcr) were present in 12 out of 29 colonies from etoposide-treated samples versus 5 out of 39 colonies from control samples (p value &lt;0.01), demonstrating that etoposide exposure promotes stable rearrangements within a hematopoietic stem cell compartment with significant proliferative potential. Eight of the 17 events were sequenced, and showed 6 MLL tandem duplications within intron 8, one complex translocation between MLL and chr.15 and tandem duplication, and one event with foreign sequence of unknown origin. Our data are the first report of the spectrum and frequency of MLL rearrangements following topo II inhibitor exposure in a cell population thought to be the target for recombinogenic events leading to therapy-related leukemias.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2617-2617
Author(s):  
Fumio Arai ◽  
Kentaro Hosokawa ◽  
Yumiko Nojima ◽  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2617 Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs. On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004). In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT. These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures: No relevant conflicts of interest to declare.

Intrinsic Molecular Features of Human Hematopoietic Stem Cells from Different Sources Define Their Specific Functional Properties

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3069-3069
Author(s):  
Maria Rosa Lidonnici ◽  
Annamaria Aprile ◽  
Marta Frittoli ◽  
Giacomo Mandelli ◽  
Ylenia Paleari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Cell Frequency ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Molecular Features ◽  
Cd34 Cells ◽  
Different Sources

Abstract Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs. So far, the intrinsic molecular features of human primitive HSCs from different sources have not been investigated in comparative studies to unravel their variable reconstitution potential. Diverse strategies are currently used to disengage HSCs from the niche, promoting egress from BM to PB. Traditionally the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, many other compounds have been tested to this regard. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the PB. This drug, which was shown in preclinical combination studies with G-CSF to enhance mobilization compared to G-CSF alone, is currently approved by FDA and EMA "in combination with G-CSF to enhance the mobilization of HSCs into the peripheral blood for collection and autologous transplantation of patients affected by lymphoma or multiple myeloma whose cells mobilize poorly" We investigated functional and molecular hallmarks of human HSCs from different sources, i.e. BM and PB following mobilization by G-CSF and/or Plerixafor. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+ CD38/low CD90+ CD45RA- CD49f+ cells and primitive populations of HSCs. These cells are able to provide stable long-term hematopoietic engraftment in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The quiescence status of these cells correlates with the enriched scid-repopulating cell frequency. Noteworthy, the combined use of G-CSF and Plerixafor mobilizes a CD34+ population enriched in immature cells and with a lower engraftment capacity respect to cells mobilized by Plerixafor alone. Since the signaling provided by the interaction of SDF-1 with CXCR4, plays an essential role in maintaining HSC quiescence and regulating homing, we analyzed the CXCR4 expression. Interestingly, this analysis reveals that the proportion of CXCR4+ primitive cells was lower when using G-CSF combined to Plerixafor in respect to Plerixafor alone. These data indicate that the combination of the two mobilizing agents induce a higher amount of circulating CD34+ cells but containing a lower proportion of cells capable of homing to BM in NSG mice. . As a result, at a defined dose of transplanted CD34+ cells, less SRCs are observed when G-CSF is added to Plerixafor. Indeed, it is expected to observe also a rapid rescue of hematopoiesis in myeloablated subjects conferred by high amount of short-term progenitors. Insights into the transcriptional program reveal the molecular machinery underlying stemness features of cells derived from different sources, defining their specific functional properties. Noteworthy, CD34+ cells exposed to Plerixafor but still resident in the BM acquire an intermediate signature between steady-state and circulating cells, suggesting an effect of this agent on HSC function. From preliminary data, genes of Prostaglandin signaling are up-regulated in HSCs mobilized by Plerixafor, suggesting a role of this pathway. These data uncover unique HSCs properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document