Bmi1 Confers Stress Resistance to Self-Renewing Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1607-1607
Author(s):  
Shunsuke Nakamura ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stress Resistance ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Ex Vivo Culture ◽  
The Impact ◽  
Serial Transplantation

Abstract Abstract 1607 The polycomb group (PcG) proteins form chromatin-modifying complexes that implement transcriptional silencing. There are two major PcG complexes, polycomb repressive complex (PRC) 1 and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Among PcG proteins, Bmi1, a core component of PRC1, plays an essential role in the self-renewal and maintenance of various kinds of stem cells including hematopoietic stem cells (HSCs), neural stem cells, and leukemic stem cells. We previously reported that forced expression of Bmi1 using a Bmi1 retrovirus promotes symmetrical cell division of HSCs, resulting in a marked ex vivo expansion of multipotent progenitors and an enhancement of repopulating capacity of HSCs in vivo. However, the impact of overexpression of Bmi1 in HSCs remains to be precisely addressed. To this end, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the Rosa promoter in a tissue-specific fashion by the Cre-LoxP system. We crossed the mice to Tie2-Cre mice (Tie2-Cre; Rosa-Bmi1fl/+) and induced overexpression of Bmi1 in hematopoietic cells in vivo. Real-time PCR analysis demonstrated that expression of Bmi1 inpurified bone marrow (BM) c-Kit+Sca-1+Lineage-marker- (KSL) cells was 6-fold higher in Tie2-Cre; Rosa-Bmi1fl/+ mice than control Tie2-Cre mice mice. Overexpression of Bmi1 did not significantly affect steady state hematopoiesis. The number of HSCs and progenitors (multipotent progenitors, CMPs, GMP, MEPs, and CLPs) and lineage composition (myeloid cells, B cells, and T cells) in BM of Tie2-Cre; Rosa-Bmi1fl/+ mice was not significantly changed compared to those in control mice. We then performed serial transplantation assay. The repopulating capacity of Tie2-Cre; Rosa-Bmi1fl/+ BM cells was comparable to those of the control cells in primary recipients. However, Tie2-Cre; Rosa-Bmi1fl/+ cells retained higher repopulating capacity during serial transplantation compared to those of the control cells. Moreover, ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs for 10 days contained approximately 2-fold more high proliferative potential colony-forming cells (HPP-CFCs; colony diameter>1mm) and colony-forming unit-neutrophil/macrophage/erythroblast/megakaryocyte (CFU-nmEM) which retain multi-lineage differentiation potential along myeloid lineage than the control. Of note, cells in ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs exhibited significantly augmented repopulating capacity in recipient mice and we are now engaged in competitive repopulation unit (CRU) assays to determine the net HSC expansion by overexpression of Bmi1 during ex vivo culture. These results indicate that overexpression of Bmi1 confers stress resistance to HSCs during ex vivo culture and serial transplantation. Our findings also provide Bmi1 as a potential target for efficient manipulation of HSCs ex vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 83-94 ◽  
Author(s):  
Terumasa Umemoto ◽  
Masayuki Yamato ◽  
Jun Ishihara ◽  
Yoshiko Shiratsuchi ◽  
Mika Utsumi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Matrix Protein ◽  
Ex Vivo ◽  
Extracellular Matrix Protein ◽  
Αvβ3 Integrin ◽  
Hematopoietic Stem ◽  
Β3 Integrin ◽  
Ex Vivo Culture

AbstractThroughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

scholarly journals α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 558
Author(s):  
Laura Rodriguez ◽  
Pascale Duchez ◽  
Nicolas Touya ◽  
Christelle Debeissat ◽  
Amélie V. Guitart ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Alpha Tocopherol ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
G0 Phase ◽  
The Impact

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors

Blood ◽  
2014 ◽  
Vol 123 (8) ◽  
pp. 1167-1177 ◽  
Author(s):  
Patricia Benveniste ◽  
Pablo Serra ◽  
Dzana Dervovic ◽  
Elaine Herer ◽  
Gisele Knowles ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Immune Reconstitution ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Key Points

Key Points Notch signals expand human HSC (CD90low) cells in vitro and delay the expansion of CD45RAint and CD45RAhi cells in vitro. HSCs expanded in vitro are equal to ex vivo CD90low cells in immune reconstitution.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Lysosomal Activation Is Required for Priming of Quiescent Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 722-722
Author(s):  
Tasleem Arif ◽  
Raymond Liang ◽  
Maio Lin ◽  
Svetlana Kalmikova ◽  
Artem Kasianov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Ex Vivo ◽  
Specific Inhibitor ◽  
Vehicle Control ◽  
Specific Marker ◽  
Hematopoietic Stem ◽  
Concanamycin A

Despite their immense in vivo repopulating capacity, hematopoietic stem cells (HSCs) are largely quiescent at the steady-state. However, mechanisms that regulate HSC quiescence/cycling remain incompletely understood. Using mitochondrial membrane potential (MMP) to dissect the heterogeneity of HSCs (LSKCD150+CD48-), we find that HSCs within 25% lowest MMP (MMP-low) fractions are almost entirely (~95% ±2.65) in G0 as measured by Pyronin Y/Hoechst staining (p<0.05, n=3). In contrast, HSCs within 25% highest MMP (MMP-high HSCs) are in majority in cycling (see abstract 129099). To elucidate mechanisms implicated in the regulation of HSC cycling at the single cell level in quiescent MMP-low versus primed MMP-high HSCs we used single-cell RNA-Seq (scRNA-Seq) analysis. Cycling analysis in silico in each cell by CYCLONE further confirmed that over 80% of MMP-low HSCs are within G0/G1, as compared to less than 40% of MMP-high HSCs that are mostly in the S/G2/M phase. Notably, GO enrichment analysis related to protein degradation through lysosomal- and proteasomal-mediated pathways were significantly enriched in MMP-low HSCs (p=0.002). Strikingly, and in agreement with our scRNA-seq analysis, a greater abundance of lysosomes was observed in MMP-low relative to -high HSCs (p=0.002). Higher expression of lysosomal genes was further confirmed by qRT-PCR in MMP-low relative to -high HSCs. Analysis of lysosomal content by immunofluorescence staining showed that while the lysosomal specific marker LAMP2 was barely detectable in MMP-high HSCs, LAMP2 was readily found in MMP-low HSCs, results further confirmed by additional markers LAMP1 and LysoTracker Green. Lysosomes are, among others, a major component of organelle degradation through autophagy, which is required for the maintenance of HSCs however, whether lysosomes are implicated in regulating HSC beyond autophagy is unknown. To address this we examined the effect of the suppression (and not activation that is required for autophagy) of lysosomal activation on in vitro HSC maintenance. Treatment with concanamycin-A (ConA), a specific inhibitor of lysosomal acidification via inhibition of the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase) led to 3 fold improved frequency of phenotypically defined HSCs from optimally cultured lineage-negative cells in 24 hours (p<0.05, n=4). This was associated with 4-fold greater retention of the MMP-low HSC fraction (p<0.05, n=4). Cell divisions of single MMP-low and -high GFP+ HSCs treated with ConA or vehicle control was tracked up to 60 hours in culture. Over 70% of control treated MMP-low GFP+ HSCs did not divide during this time, whereas the majority (>85%) of MMP-high GFP+ HSCs divided at least once (p=0.001, n=5). While ConA treatment had only a slight effect on non-dividing MMP-low HSCs in culture, it significantly increased the frequency of non-dividing MMP-high GFP+ HSCs (p=0.007). Priming of MMP-low to -high HSCs was associated with lysosomal recruitment, and activation of mTOR signaling in MMP-high HSCs (p=0.001, n=5). Importantly, ConA-treatment led to the repression of mTOR expression and activity in MMP-high HSCs (p<0.001). In addition, a 48-hours ConA treatment led to enhanced frequency of LTC-ICs recovered in limiting dilution analysis of both MMP-low (p=0.023) and -high (p=0.004) HSCs ex vivo. To further investigate the role of suppression of lysosomal activation in vivo, FACS-purified MMP-low and -high HSCs were treated with vehicle control or ConA ex vivo for 4 days before 50 ConA- or control-treated MMP-low or -high HSCs were mixed with CD45.2 (2x105) competitor cells and injected into lethally irradiated mice (n=7) in a competitive repopulation assay. Reconstitution levels were consistently more robust in ConA-treated populations of MMP-low (p= 0.001) and -high (p=0.001) HSCs after 18 weeks as compared to control. Importantly, HSC-derived lineage output was balanced in its composition up to 18 weeks in recipients of MMP-low HSC regardless of ConA treatment as well as in ConA-treated MMP-high HSCs, while control MMP-high HSC was myeloid-biased. Overall our results, based on HSC mitochondrial heterogeneity, suggest that lysosomal -content and activity participate in the maintenance of HSC quiescence. Based on these findings, we propose a model that stipulates that lysosomal activation primes HSCs (G0⇒G1) while lysosomal suppression maintains HSC quiescence. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

scholarly journals Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

2019 ◽  
Vol 3 (15) ◽  
pp. 2355-2367 ◽  
Author(s):  
Yueh-Chwen Hsu ◽  
Tsung-Chih Chen ◽  
Chien-Chin Lin ◽  
Chang-Tsu Yuan ◽  
Chia-Lang Hsu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Knockout Mice ◽  
Neurodevelopmental Disorder ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
The Impact

Abstract Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

Foamy Viral Vectors Efficiently Transduce Quiescent Hematopoietic Stem/Progenitor Cells (HSC) and Restore the Long Term Repopulating Activity of Fancc −/− Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 182-182 ◽  
Author(s):  
D. Wade Clapp
Keyword(s):  
Stem Cells ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Foamy Virus ◽  
Reporter Construct ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Increased Risk ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is characterized by bone marrow aplasia and myeloid leukemia. The identification of FA genes raises the potential of using gene transfer technology to introduce cDNAs into autologous HSCs. Current strategies using Moloney retroviruses require a 2–4 day ex vivo culture of HSC to facilitate stable integration of the transgene. However, ex vivo culture results in a time-dependent increase in apoptosis of Fancc−/− primitive HSC and mice reconstituted with the surviving cells have an increased risk of acquiring myeloid malignancies. Therefore we examined the potential of a recombinant foamy virus construct (MD9-FANCC-EGFP) to transduce murine Fancc −/− HSC in the absence of prestimulation. Forty-80% of progenitors that were in G0 – G1 at the time of transduction were transduced following a single 10–14 hr transduction. Aliquots of MD9-FANCC-EGFP transduced BM cells or cells encoding the EGFP transgene only were transplanted into irradiated recipient mice or recipients treated with IFN-g only. Four-six months following transplantation, recipient BM cells were isolated and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. Fancc−/− progenitors encoding recombinant FANCC were found to have a similar resistance to MMC as wildtype (WT) controls while Fancc−/− progenitors encoding the reporter construct only retained a high sensitivity to MMC. To assess the potential of MD9-FANCC-EGFP to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. The repopulating activity of MD9-FANCC-EGFP-transduced Fancc−/− stem cells was comparable to WT controls 18–24 months following transplantation in primary and secondary recipients. Additionally, while mice reconstituted with Fancc−/− cells transduced with the reporter construct had reduced repopulating ability as compared to the other groups, none of these recipients acquired myeloid malignancies. Collectively, these data provide in vivo evidence that an abbreviated transduction protocol utilizing a foamy-viral based vector allows efficient transduction of Fancc−/− HSC, and diminishes the selection pressure that occurs during ex vivo culture of Fancc−/− HSCs.

In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia

Blood ◽  
1999 ◽  
Vol 94 (6) ◽  
pp. 2151-2158 ◽  
Author(s):  
Kevin P. Battaile ◽  
Raynard L. Bateman ◽  
Derik Mortimer ◽  
Jean Mulcahy ◽  
R. Keaney Rathbun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Serial Transplantation

Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.

Sign in / Sign up

Export Citation Format

Share Document