Co-Transplantation of MSC Improves the Engraftment of HSC after Auto-iBMT in Non-Human Primates.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2321-2321
Author(s):  
Shigeo Masuda ◽  
Yoko Obara ◽  
Naohide Ageyama ◽  
Hiroaki Shibata ◽  
Tamako Ikeda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Osteoblastic Differentiation ◽  
The Body ◽  
The Other ◽  
Hematopoietic Stem ◽  
Cynomolgus Monkeys ◽  
Colony Pcr ◽  
Marrow Cavity

Abstract [Background] Mesenchymal stem cells (MSC) have been shown to play critical roles in various in vivo phenomena including osteoblastic differentiation. It has been suggested that, in the bone marrow, hematopoietic stem cells (HSC) reside in osteoblastic niche, which consists of osteoblasts derived from MSC. In mice, previous studies have demonstrated that co-transplantation of MSC improves the engraftment of HSC, especially after transplantation of the cells into the bone marrow cavity directly, namely intra-bone marrow transplantation (iBMT). However, neither the efficacy nor the dynamics such as migration and homing of HSC after iBMT with MSC have been determined in large animals. Here, using non-human primates, we have investigated the effects of co-transplantation of MSC on the engraftment of HSC after autologus iBMT. [Methods] Auto-iBMT of cynomolgus monkeys was performed, using bone marrow stromal cells (as MSC) and CD34-positive cells (as HSC). The latter were divided into two equal aliquots, each of which was genetically marked with a different retroviral vector, G1Na or LNL6. Conditioning of iBMT, TBI or administration of busulfan, was followed by hemi-iBMT; that is, the bone marrow of one side (right or left) of the body was transplanted with one HSC aliquot together with MSC, whereas the other side of the identical body was transplanted with the other HSC aliquot alone. Engraftment of each HSC aliquot was evaluated by colony PCR of bone marrow, as well as by PCR of the genomic DNA obtained from peripheral blood or bone marrow of humerus, femur, and ilium. Both PCR could distinguish the dual markings derived from the two HSC aliquots. [Results] In the first monkey transplanted, we found that the engraftment derived from the co-transplantation aliquot was 4.4-times higher than that derived from the HSC alone aliquot as assessed by colony PCR (48% versus 11%) using the bone marrow samples obtained from the ilium at day 46 post-iBMT. In the second monkey, when the peripheral WBC recovered to 2500–3000/μl after day 28 post-iBMT, 2% of the cells were positive with the retroviral marking derived from the co-transplantation aliquot, although none of them were positive with that derived from the HSC alone aliquot. In addition, colony PCR of the humerus and femur of both sides at day 39 post-iBMT revealed that the engraftment derived from the co-transplantation aliquot was 6.0-times higher than that derived from the HSC alone aliquot. Notably, colony-forming units (CFU) derived from the cotransplantation aliquot were detected in the bone marrow of the opposite side, suggesting that HSC injected into the bone marrow might migrate and achieve homing in the distant bone marrow. [Conclusion] Taken together, these results indicate that, in auto-iBMT of cynomolgus monkeys, co-transplantation of MSC improves the engraftment of HSC, the efficacy of which might be attributable to additional osteoblastic niche, presumably created from co-transplanted MSC.

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

1987 ◽  
Vol 5 (3) ◽  
pp. 231-241 ◽  
Author(s):  
Vincent S. Gallicchio ◽  
Thomas D. Watts ◽  
George P. Casale ◽  
Philip M. Bartholomew
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Hematopoietic Stem

scholarly journals The Act of Controlling Adult Stem Cell Dynamics: Insights from Animal Models

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 667
Author(s):  
Meera Krishnan ◽  
Sahil Kumar ◽  
Luis Johnson Kangale ◽  
Eric Ghigo ◽  
Prasad Abnave
Keyword(s):  
Stem Cells ◽  
Animal Models ◽  
Adult Stem Cells ◽  
The Body ◽  
In Vivo Studies ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Organ Systems

Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs).

scholarly journals Syngeneic bone marrow transplantation without conditioning in a patient with paroxysmal nocturnal hemoglobinuria: in vivo evidence that the mutant stem cells have a survival advantage

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 742-750 ◽  
Author(s):  
M Endo ◽  
PG Beatty ◽  
TM Vreeke ◽  
CT Wittwer ◽  
SP Singh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Pathological Process ◽  
Survival Advantage ◽  
Hematopoietic Stem ◽  
Glycosyl Phosphatidylinositol ◽  
Normal Expression ◽  
Marrow Infusion

A 10-year-old girl with paroxysmal nocturnal hemoglobinuria (PNH) received an infusion of syngeneic bone marrow without preparative marrow ablation or immunosuppression. Following transplant, the patient became asymptomatic in concordance with an increase in the percentage of peripheral blood cells with normal expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). However, molecular analysis suggested engraftment of a relatively small number of donor stem cells and persistence of an abnormal stem cell with mutant PIG-A. During 17 months of observation, the percentage of cells with normal GPI-AP expression gradually decreased, while intravascular hemolysis progressively increased. Approximately 16.5 months post-transplant, the patient once again became symptomatic. Together, these results indicate that syngeneic marrow infusion provided a clinical benefit by increasing the proportion of erythrocytes with normal expression of GPI- anchored complement regulatory proteins without supplanting the abnormal stem cells. However, evidence of insidious disease progression following the marrow infusion implies that the abnormal stem cells have a survival advantage relative to the transplanted stem cells. Thus, these studies contribute in vivo data in support of the hypothesis that PNH arises as a consequence of a pathological process that selects for hematopoietic stem cells that are GPI-AP-deficient.

scholarly journals Direct evidence that natural killer cells in nonimmune spleen cell populations prevent tumor growth in vivo

1979 ◽  
Vol 149 (5) ◽  
pp. 1260-1264 ◽  
Author(s):  
M Kasai ◽  
JC Leclerc ◽  
L McVay-Boudreau ◽  
FW Shen ◽  
H Cantor
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Tumor Growth ◽  
Nk Cells ◽  
Spleen Cell ◽  
Cell Population ◽  
The Body ◽  
Nk Activity

Relatively large numbers of nonimmune spleen cells do not protect against the local growth of two lymphomas. However, this heterogeneous population of splenic lymphocytes contains a subset of cells that efficiently protects against in vivo tumor growth. This cell population (cell-surface phenotype Thyl.2(-)Ig(-)Ly5.1(+)) represents less than 5 percent of the spleen cell population and is responsible for in vitro NK-mediated lysis. Although these studies clearly and directly demonstrate that Ly5(+) NK cells selected from a heterogeneous lymphoid population from nonimmune mice can protect syngeneic mice against local in vivo growth of two different types of tumor cells (in contrast to other lymphocyte sets within the spleen), they do not directly bear upon the role of NK cells in immunosurveillance. They do indicate that highly enriched Ig(-)Thyl(-)Ly5(+) cells, which account for virtually all in vitro NK activity, can retard tumor growth in vivo. It is difficult to ascribe all anti-tumor surveillance activity to NK cells, because they probably do not recirculate freely throughout the various organ systems of the body. Perhaps NK ceils may play a role in prevention of neoplastic growth within discrete anatomic compartments where there is rapid differentiation of stem cells to mature progeny (e.g., bone marrow, spleen, and portions of the gastrointestinal tract)and may normally act to regulate the growth and differentiation of non-neoplastic stem cells. Long-term observation of chimeric mice repopulated with bone marrow from congenic or mutant donors expressing very low or very high NK activity may help to answer these questions.

Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3737-3747 ◽  
Author(s):  
Dirk Heckl ◽  
Daniel C. Wicke ◽  
Martijn H. Brugman ◽  
Johann Meyer ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Egfp Reporter

AbstractThpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl−/−) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl−/− bone marrow cells into Mpl−/− mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl−/− cells had increased long-term repopulating potential, with a marked increase in lineage−Sca1+cKit+ cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage−Sca1+cKit+ cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Long-Term In Vivo Expression from a Self-Inactivating Lentiviral Vector Flanked by a Chromatin Insulator Element.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1289-1289
Author(s):  
Ping Xia ◽  
Richard Emmanuel ◽  
Kuo Isabel ◽  
Malik Punam
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Lentiviral Vector ◽  
Gfp Expression ◽  
Hematopoietic Stem ◽  
Insulator Element ◽  
Gfp Fluorescence

Abstract We have previously shown that self-inactivating lentiviral vectors infect quiescent hematopoietic stem cells (HSC), express long-term, resist proviral silencing in HSC and express in a lineage specific manner. However, their random integration into the host chromosome results in variable expression, dependent upon the flanking host chromatin (Mohamedali et al, Mol. Therapy 2004). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated’ from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector in the RBC progeny of hematopoietic stem cells (HSC) in long term in vivo. We designed an erythroid-specific SIN-lentiviral vector I8HKGW, expressing GFP driven by the human ankyrin gene promoter and containing two erythroid-specific enhancer elements and compared it to an analogous vector I8HKGW-I, where the cHS4 insulator was inserted in the SIN deletion to flank the I8HKGW expression cassette at both ends upon integration. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency and GFP+ cells were sorted to generate clones. Single copy MEL clones showed no difference in the mean GFP fluorescence intensity (MFI) between the I8HKGW+ and the I8HKGW-I+ MEL clones. However, there was a reduction in the chromatin position effect variegation (PEV), reflected by reduced coefficient of variation of GFP expression (CV) in I8HKGW-I clones (n=115; P<0.01), similar to in vitro results reported by Ramezani et al (Blood 2003). Next, we examined for expression and PEV in the RBC progeny of HSC, using the secondary murine bone marrow transplant model. Lethally irradiated C57Bl6 (CD45.2) mice were transplanted with I8HKGW and I8HKGW-I transduced B6SJL (CD45.1) Sca+Lin- HSC and 4–6 months later, secondary transplants were performed. Mice were analyzed 3–4 months following secondary transplants (n=43). While expression from both I8HKGW and I8HKGW-I vectors appeared similar in secondary mice (46±6.0% vs. 48±3.6% GFP+ RBC; MFI 31±2.6 vs. 29±1.4), there were 0.37 vs. 0.22 copies/cell in I8HKGW and I8HKGW-I secondary recipients, respectively (n=43), suggesting that the probability of GFP expression from I8HKGW-I vectors was superior when equalized for vector copy. The CV of GFP fluorescence in RBC was remarkably reduced to 55±1.7 in I8HKGW-I vs. 196±32 in I8HKGW RBC (P<0.001). We therefore, analyzed these data at a clonal level in secondary CFU-S and tertiary CFU-S. The I8HKGW-I secondary CFU-S had more GFP+ cells (32.4±4.4%) vs. I8HKGW CFU-S (8.1±1.2%, n=143, P<0.1x10E-11). Similarly, I8HKGW-I tertiary CFU-S also had more GFP+ cells (25±1.8%) vs. I8HKGW CFU-S (6.3±0.8%, n=166, P<0.3x10E-10). We also plated bone marrow from secondary mice in methylcellulose and analyzed GFP expression in individual BFU-E. The I8HKGW-I tertiary BFU-E had more GFP+ cells (28±3.9%) vs. I8HKGW BFU-E (11±5%, n=50, P<0.03) with significantly reduced CV (67 vs 125, n=50, P<6.6X10E-7). Taken together, the ‘insulated’ erythroid-specific SIN-lentiviral vector increased the probability of expression of proviral integrants and reduced PEV in vivo, resulting in higher, consistent transgene expression in the erythroid cell progeny of HSC. In addition, the enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these vectors for gene therapy for RBC disorders.

Prostaglandin E2 (PGE2) Regulates Osteoblastic Jagged1 and Expands Primitive Hematopoietic Cells In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 89-89 ◽  
Author(s):  
Laura M. Calvi ◽  
Benjamin J. Frisch ◽  
Benjamin J. Gigliotti ◽  
Christina A. Christianson ◽  
Jonathan M. Weber ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cortical Bone ◽  
Osteoblastic Cells ◽  
Pcr Analysis ◽  
Inhibitory Peptide ◽  
Hematopoietic Stem ◽  
Hsc Niche

Abstract Parathyroid Hormone (PTH) targets osteoblastic cells (OBs) in the bone marrow microenvironment and expands hematopoietic stem cells (HSC) through Notch activation. Since PTH stimulates the Notch ligand Jagged1 (J1) in OBs, we have focused on the signaling pathways involved in this PTH effect in order to identify novel activators of the HSC niche. Osteoblastic Protein Kinase A (PKA) activation is required for the PTH-dependent J1 increase in OBs. Therefore, we hypothesized that alternative PKA activators could also regulate osteoblastic J1, alter the HSC niche, and provide additional pharmacologic tools to expand HSC in vivo. Consistent with this hypothesis, direct PKA agonists 8-bromo-cAMP and dibutyryl-cAMP stimulated J1 in osteoblastic UMR106 cells. In addition, PGE2, a member of the prostaglandin family known to stimulate PKA in OBs, was studied in vivo and in vitro. By real-time RT-PCR analysis, J1 mRNA was increased up to 5 fold at 2 hours in UMR106 cells when treated with PGE2 (10−7 M) compared to vehicle. J1 protein was also increased after treatment with PGE2. The PGE2-dependent J1 increase was blocked in the presence of the specific PKA inhibitors H89 and myristoylated PKA Inhibitory Peptide (14–22)(PKI) (200ug/ml), demonstrating that PKA is necessary for osteoblastic J1 stimulation by PGE2. Since systemic PGE2 is known to have bone anabolic effects in both humans and animal models, adult wild-type FVB/N male mice were treated with PGE2 (6mg/kg/day i.p.) for 12 days. This regimen has previously been shown to have bone anabolic effects in rats. At day 12, histologic analysis demonstrated an anabolic effect mainly on cortical bone, as was evident in the femurs and tibiae of PGE2-treated mice compared to control. This histologic finding was confirmed by histomorphometry (trabecular bone area means 41% vs 12%,p=0.0916, n=3 in both groups; cortical thickness means 138 vs 85 μm, p=0.0071, n=3 in both groups). Frequency of hematopoietic stem cells (c-Kit+, Sca1+, lin−) was increased in bone marrow from PGE2-treated vs control mice by over 20% (p=0.0018, n=8 in both groups). In summary, PGE2 stimulates J1 in osteoblastic cells through PKA activation and increases mainly cortical bone in vivo. Ongoing studies will confirm whether in vivo PGE2 treatment expands HSC, and whether osteoblastic J1 regulates this process. This study identifies PGE2 as a novel regulator of osteoblastic J1, and as a potential new microenvironmental modulator of HSC, which could be used for in vivo therapeutic HSC niche manipulation.

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