Mutant ASXL1 Promotes Expansion of the Phenotypic Hematopoietic Stem Cell Compartment

Somatic mutations of the ASXL1 gene are recurrently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations causes CH are not understood. Here, using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we investigated the effect of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). 　To examine the influence of ASXL1-MT on hematopoiesis, we bred the ASXL1-MT-KI mice with Vav-Cre transgenic mice. Young ASXL1-MT-KI mice (6-12 weeks) did not show significant changes in hematological parameters and differentiation status of peripheral blood. We observed the decreased frequency of hematopoietic stem and progenitor cells (HSPCs), including long-term HSCs (LT-HSCs). Competitive transplantation assays showed the reduced repopulation ability in ASXL1-MT-KI HSPCs. Thus, ASXL1-MT decreased the number and impaired the function of HSPCs in young mice. 　Next, we examined age-related changes in hematopoiesis caused by ASXL1-MT. Aged ASXL1-MT-KI mice displayed a myeloid-biased differentiation and hypocellular bone marrow, indicating the dysfunction of hematopoiesis. Interestingly, ASXL1-MT markedly increased the frequency of phenotypic LT-HSCs (pLT-HSCs) in aged mice (20-24 months). Competitive transplantation assays showed the impaired repopulation potential of pLT-HSCs from aged ASXL1-MT-KI mice. These data demonstrate that the increased pLT-HSCs in aged ASXL1-MT-KI mice are not functional HSCs with long-term repopulation potential. 　To elucidate how ASXL1-MT drives HSPC dysfunction, we conducted RNA-Seq analysis using HSPCs from young mice. This analysis revealed upregulation of mitochondrial genes in ASXL1-MT-KI HSPCs. In addition, MitoTracker staining, extracellular flux analyses and metabolome analyses demonstrated the enhanced mitochondrial metabolism in ASXL1-MT-KI HSPCs. We also found that the aberrantly elevated mitochondrial activity induced ROS overproduction and increased DNA damage, resulting in HSPC dysfunction. 　As a mechanism underlying the enhanced mitochondrial activity of ASXK1-MT-KI HSPCs, we revealed that ASXL1-MT activated the Akt/mTOR pathway in HSPCs. Treatment with an Akt inhibitor perifosine or an mTOR inhibitor rapamycin normalized the mitochondrial membrane potential and ROS levels in ASXL1-MT-KI HSPCs. Moreover, rapamycin treatment improved engraftment of ASXL1-MT-KI bone marrow cells after transplantation. These data indicate that the activated Akt/mTOR signaling leads to the enhanced mitochondrial activity, elevated ROS levels, and HSPC dysfunction in ASXL1-MT-KI mice. 　To assess the impact of the enhanced Akt/mTOR signaling on age-related changes in ASXL1-MT-KI mice, we administered rapamycin to aged ASXL1-MT-KI mice. Intriguingly, rapamycin treatment decreased the frequency of pLT-HSCs, and normalized the bone marrow cellularity in aged ASXL1-MT-KI mice. Cell cycle analysis revealed that pLT-HSCs in G0 phase were decreased in aged ASXL1-MT-KI mice, which was normalized by rapamycin treatment. These data demonstrate that the activated Akt/mTOR pathway provokes the aberrant expansion of pLT-HSCs in aged ASXL1-MT-KI mice. 　We next attempted to clarify the underlying mechanism of Akt activation in ASXL1-MT-KI mice. Immunoprecipitation experiments revealed that ASXL1-MT/BAP1 complex deubiquitinated AKT in 293T cells. To determine the role of endogenous Bap1 on Akt signaling, we assessed the effect of Bap1 deletion in murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells). A time course experiments showed Akt phosphorylation induced by IL-3 stimulation was attenuated and shortened in Bap1-depleted cSAM cells. These data suggest that ASXL1-MT/BAP1 complex deubiquitinate and stabilize phosphorylated Akt. 　In summary, we demonstrated that ASXL1-MT cooperated with BAP1 to promote AKT deubiquitination and activation. The activated Akt/mTOR pathway led to enhanced mitochondrial metabolism, elevated ROS levels and increased DNA damage. These molecular bases underlie the age-associated expansion of the pLT-HSC compartment. Our results underscore the possibility that CH can originate from a pLT-HSC with a limited repopulation potential. A pharmacological inhibition of the Akt/mTOR pathway could be a promising therapeutic intervention to individuals with CH harboring ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

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NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Blood ◽

10.1182/blood.v126.23.1155.1155 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1155-1155

Author(s):

Stefanie Kreutmair ◽

Rouzanna Istvanffy ◽

Cathrin Klingeberg ◽

Christine Dierks ◽

Christian Peschel ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Stress Response ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Hematopoietic Stem ◽

Age Related

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days). To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response. To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

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Loss of Dnmt3a Confers Resistance to Pegifnα in JAK2-V617F Mouse Model

Blood ◽

10.1182/blood-2020-140548 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Jan Stetka ◽

Nils Hansen ◽

Lucia Kubovcakova ◽

Hui Hao-Shen ◽

Stefan Dirnhofer ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Double Mutant ◽

Bone Marrow Cells ◽

Jak2 V617f ◽

Mutant Mice ◽

Chimeric Mice ◽

Hematopoietic Stem ◽

Marrow Cells

Myeloproliferative neoplasms (MPNs) are clonal hematological malignancies, which are initiated and maintained by a pool of malignant long-term hematopoietic stem cells (LT-HSCs). A recurrent gain-of-function mutation in JAK2-V617F (VF) is present in a majority of patients with MPN. Treatment with pegylated interferon alpha (pegIFNα) is currently the only therapy known to reduce JAK2-V617F+ LT-HSCs and to induce a complete molecular remission (CMR) in a subset of patients. Long-term follow-up studies suggested, that patients with additional mutations in epigenetic modifiers, such as DNMT3A, have poorer responses to pegIFNα and are less likely to achieve CMR. We therefore hypothesized that DNMT3A loss leads to alterations in JAK2-V617F HSCs functions conferring resistance to pegIFNα treatment. To test the hypothesis we performed competitive transplantations of bone marrow cells from VF or VF;Dnmt3a-/- mice, which also co-express the GFP reporter mixed with wildtype competitor (WT) cells in 1:20 ratio (Figure 1A). After 6 weeks, recipients mice were randomized and treated for 16 weeks with mouse pegIFNα (25µg/kg; s.c.; once per week) or saline (Figure 1A). Complete blood counts and GFP chimerism monitored every 4 weeks showed, that pegIFNα was able to achieve normalization of peripheral blood parameters and reduce GFP chimerism in both, VF and VF;Dnmt3a-/- chimeric mice. In VF chimeric mice treated with pegIFNα we observed a significant reduction of splenomegaly (Figure 1B) and normalization of bone marrow MPN histopathology compared to saline, however pegIFNα treatment in double-mutant VF;Dnmt3a-/- did not improve histopathological features and had tendency to worsen splenomegaly. GFP chimerism in LT-HSCs and committed hematopoietic progenitors was reduced only in VF chimeric mice, but not in double mutant mice. Chronic activation and ensuing replication-induced DNA damage is a critical factor for the functional decline and depletion of mutant LT-HSCs upon treatment with pegIFNα. Our functional analysis shows that LT-HSCs and early progenitors from double-mutant VF;Dnmt3a-/- mice were less prone to accumulate reactive oxygen species, DNA damage and exit dormancy upon treatment with pegIFNα, compared to VF (Figure 1B). In series of secondary transplantations at increasingly limiting dilutions, bone marrow cells from VF mice treated with pegIFNα showed reduced repopulation capacity resulting in a milder MPN phenotype and lower mutant LT-HSC chimerism in the secondary recipients. In contrast, bone marrow cells from VF;Dnmt3a-/- double mutant mice treated with pegIFNα gave rise to a more aggressive disease phenotype compared to saline in secondary (Figure 1C) and also tertiary transplantations (not shown). Recipients transplanted with BM cells from VF;Dnmt3a-/- mice treated with pegIFNα also showed increased frequencies of mutant LT-HSCs compared to saline. Our results suggest that the accelerating effects of pegIFNα therapy inflicted on the LT-HSC of VF;Dnmt3a-/- double mutant mice are stably inherited upon secondary and tertiary transplantations. Overall, our results demonstrate that loss of Dnmt3a mitigate functional attrition of JAK2-V617F+ LT-HSCs upon chronic pegIFNα treatment and create vastly expanded pool of resistant LT-HSCs and point towards possible detrimental effects of pegIFNα therapy in patients with additional loss-of-function mutation in DNMT3A. Disclosures No relevant conflicts of interest to declare.

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CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽

10.1182/blood.v87.10.4136.bloodjournal87104136 ◽

1996 ◽

Vol 87 (10) ◽

pp. 4136-4142 ◽

Cited By ~ 113

Author(s):

I Kawashima ◽

ED Zanjani ◽

G Almaida-Porada ◽

AW Flake ◽

H Zeng ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Marrow Cell ◽

In Utero ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Show Evidence ◽

Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

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Age related changes in hemopoietic capacity of bone marrow cells

Mechanisms of Ageing and Development ◽

10.1016/0047-6374(89)90029-8 ◽

1989 ◽

Vol 48 (1) ◽

pp. 91-99 ◽

Cited By ~ 27

Author(s):

A. Sharp ◽

D. Zipori ◽

J. Toledo ◽

S. Tal ◽

P. Resnitzky ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Age Related ◽

Age Related Changes ◽

Marrow Cells

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Normal cycling patterns of hematopoietic stem cell subpopulations: an assay using long-term in vivo BrdU infusion

Blood ◽

10.1182/blood.v66.6.1460.bloodjournal6661460 ◽

1985 ◽

Vol 66 (6) ◽

pp. 1460-1462 ◽

Cited By ~ 4

Author(s):

ME Pietrzyk ◽

GV Priestley ◽

NS Wolf

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Improved Method ◽

Hematopoietic Stem ◽

Infusion Study ◽

A Cell ◽

Cell Subpopulations ◽

Over Time

It was found in a long-term bromodeoxyuridine (BrdU) infusion study that two or more different subpopulations of bone marrow stem cells exist in mice. One of these subpopulations appears to be noncycling and forms approximately 10% of eight-day CFU-S. Another one, a subpopulation of slowly cycling bone marrow cells, is represented as 14- day CFU-S. The 14-day CFU-S have a regular increment in the percentage of the subpopulation entering the cycle over time, with a cell generation half-time of 21 days. The cycling status in these experiments was ascertained by in vivo continuous long-term BrdU infusion. An improved method is presented for long-term BrdU infusion with UV killing of cycled cells.

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Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽

10.1182/blood.v83.4.939.939 ◽

1994 ◽

Vol 83 (4) ◽

pp. 939-948 ◽

Cited By ~ 116

Author(s):

Y Tomita ◽

DH Sachs ◽

M Sykes

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Whole Body ◽

Mixed Chimerism ◽

Hematopoietic Stem ◽

Donor Cells ◽

Donor Type ◽

Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

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Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17165865 ◽

2020 ◽

Vol 17 (16) ◽

pp. 5865

Author(s):

Ramya Dewi Mathialagan ◽

Zariyantey Abd Hamid ◽

Qing Min Ng ◽

Nor Fadilah Rajab ◽

Salwati Shuib ◽

...

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Protein Carbonyls ◽

Mouse Bone Marrow ◽

Tail Moment ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

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Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

Author(s):

SJ Szilvassy ◽

S Cory

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Bone Marrow Cells ◽

High Titer ◽

Marker Gene ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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Hmgb3 Deficiency Inhibits Down-Regulation of c-kit in Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.372.372 ◽

2004 ◽

Vol 104 (11) ◽

pp. 372-372

Author(s):

Michael J. Nemeth ◽

Stacie M. Anderson ◽

Lisa J. Garrett-Beal ◽

David M. Bodine

Keyword(s):

Bone Marrow ◽

Hind Limb ◽

Bone Marrow Cells ◽

Es Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Bone Marrow Cellularity ◽

Marrow Cellularity ◽

Marrow Cells

Abstract Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is expressed in HSC-enriched lin−, c-kitHI, Sca-1HI, IL-7Rα− (KSIL) cells and Ter119+ erythroid cells. To define Hmgb3 function, we generated hemizygous mice (Hmgb3−/Y) using 129/SvJ ES cells. Hmgb3−/Y mice contain normal numbers of KSIL cells that are capable of normal repopulation and self-renewal. However, these mice have 1.6-fold fewer common lymphoid progenitors (CLP) and 3-fold fewer common myeloid progenitors (CMP) (p < 0.05). We hypothesized that the role of Hmgb3 in early hematopoiesis involves c-kit regulation. We observed that the level of c-kit mRNA in Hmgb3−/Y HSCs increased 30% compared to wild-type (WT) (p = 0.05). We used 5-fluorouracil (5-FU), which has been shown to down-regulate c-kit on HSCs, to characterize the interaction between Hmgb3 and c-kit. We monitored Hmgb3 expression in KSIL and lin−, Sca-1+, c-kit− cells before and after 5-FU treatment (150 mg/kg) using phenotypically normal transgenic mice containing an IRES-GFP cassette knocked into the 3′ UTR of Hmgb3. Prior to 5-FU treatment, 27% of KSIL cells were GFP+ (these cells were absent 4 days post-injection {p.i.}). In contrast, 1.8% of lin−, c-kit−, Sca-1+ cells were GFP+ before 5-FU treatment whereas 26% of lin−, c-kit−, Sca-1+ cells were GFP+ 4 days p.i. The increased proportion of GFP+ lin-, c-kit−, Sca-1+ cells after 5-FU treatment is consistent with previous findings that repopulating activity resides within the c-kit−/LO population in 5-FU treated bone marrow and our finding that Hmgb3 serves as a marker for long-term repopulating activity. To determine the time course of c-kit regulation, we compared bone marrow from 5-FU injected Hmgb3−/Y and WT mice for analysis at 2, 4, and 6 days p.i. Two days p.i., both WT and Hmgb3−/Y mice contained similar numbers of bone marrow cells (7 x 106 cells/hind limb) and the KSIL population was absent. By four days p.i., the bone marrow cellularity of WT mice declined to 5.5 ± 0.9 x 106 cells/hind limb and KSIL cells were still absent. However, in Hmgb3−/Y mice 4 days p.i., bone marrow cellularity stabilized at 7.9 ± 0.8 x 106 cells/hind limb, an increase of 43% compared to WT (p < 0.01), along with the re-emergence of the KSIL population. To determine whether the Hmgb3−/Y lin−, c-kit−, Sca-1+ population contains repopulating HSCs after 4 days of 5-FU treatment similar to WT mice, we performed repopulation assays using KSIL and lin−, c-kit−, Sca-1+ cells sorted from 4 day p.i. 5-FU treated Hmgb3−/Y mice. Recipients received either 2 x 104 KSIL or 2 x 105 lin−, c-kit−, Sca-1+ cells (Ly 5.2) from 5-FU treated Hmgb3−/Y mice along with a radioprotective dose of 3 x 105 congenic (Ly 5.1) bone marrow cells. FACS analysis performed on control recipients transplanted with congenic marrow exhibited < 1% Ly 5.2 cells in the bone marrow 16 weeks after transplant. Pre-5-FU treatment, 88% of bone marrow cells were donor derived in recipients of Hmgb3−/Y KSIL cells. There was no detectable engraftment of Hmgb3−Y lin−, c-kit−, Sca-1+ cells. In contrast to WT mice, both KSIL and lin−, c-kit−, Sca-1+ cells from 5-FU treated Hmgb3−/Y mice were capable of long-term repopulation (62–82% donor derived cells). We conclude that Hmgb3 deficiency facilitates the reemergence of c-kitHI HSCs following 5-FU treatment. Mechanisms involving either enhanced HSC self-renewal or delayed differentiation into CLPs and CMPs are both consistent with our results.

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