EZH2 Mutations Are Drivers of Clonal Hematopoiesis and Leukemic Transformation in a Mouse Model of Primary Myelofibrosis

Abstract Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by aberrant myeloid differentiation, associated with disruption of the bone marrow niche with subsequent fibrosis development and a high risk of leukemic transformation. The phenotypical complexity observed in PMF likely reflects the heterogeneous mutation profile of the neoplastic stem cells driving the disease. In our former work, we identified a CD133+ hematopoietic stem / progenitor cell (HSPC) population from patient peripheral blood that can drive major PMF morbidity parameters in a xenotransplantation mouse model. Mutational analysis of the JAK2 locus at the single cell level within the CD133+ population showed highly variable levels of cells with a JAK2+/+, JAK2V617F/+, or JAK2V617F/V617F genotype, indicating that clonality is unlikely driven by JAK2 mutations. In two of these patient samples, and in a third patient sample with CALR-fs* mutations, we identified a high load of missense mutations in EZH2 (45 to 95%), suggesting they may be critical for the clonal expansion of the neoplastic stem cell compartment. EZH2 mutations are found in circa 7% of PMF patients and are correlated with poor prognosis. EZH2 is a critical enzymatic subunit of the Polycomb Repressor Complex 2, which initiates gene repression of select genes through its intrinsic activity for methylating lysine-27 of histone H3 (H3K27). To date, the exact contribution of EZH2 mutations to PMF evolution or AML transition has not been clarified. CD133+ HSPC carrying EZH2 mutations either with JAK2 or CALR mutations were transplanted into immunodeficient NOD-scid-gamma (NSG) mice. Mice engrafted with patient samples carrying either EZH2-Y633C and JAK2-V617F or EZH2-Y733* and CALR-fs* mutations showed a strikingly similar phenotype, including high human cell engraftment (10-20%), skewed myelopoiesis, dysplastic human megakaryocytes, splenomegaly, anemia, and fibrosis in either the BM or spleen. In the case of xenotransplanted mice receiving CD133+ cells with a low JAK2 burden and EZH2-D265H mutations, we observed the highest engraftment in our mouse model (62-95%) and in one case AML transition with >50% CD133+ human blasts in murine bone marrow. Notably, AML arose from a CD133+ EZH2D265H/+ cell that lacked JAK2V617Fmutation. We thus conclude that EZH2 mutations confer to CD133+ neoplastic stem cells a predisposition to clonal aberrant hematopoiesis; whereas acquisition of JAK2V617F or CALR mutations likely leads to the observed myeloproliferation and disruption of megakaryocytic and erythroid regulation . Moreover, our results demonstrate that epigenetic mutations (like EZH2D265) and not JAK2V617F are critical for AML transition. Our data underscore the importance of post-transcriptional modifiers of histones in altering the epigenetic landscape of neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones. Disclosures No relevant conflicts of interest to declare.

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Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽

10.1182/blood.v120.21.1234.1234 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1234-1234

Author(s):

Robert S Welner ◽

Giovanni Amabile ◽

Deepak Bararia ◽

Philipp B. Staber ◽

Akos G. Czibere ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cells ◽

Quiescent State ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽

10.1182/blood-2020-137026 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Yang Mei ◽

Yijie Liu ◽

Xu Han ◽

Jing Yang ◽

Peng Ji

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Inflammatory Cytokines ◽

Conflicts Of Interest ◽

Double Knockout ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Cell Counts ◽

Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

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MT1-MMP Regulates Hematopoiesis Through HIF-Mediated Chemo-/Cytokine Release From the Bone Marrow Niche,

Blood ◽

10.1182/blood.v118.21.3409.3409 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3409-3409

Author(s):

Chiemi Nishida ◽

Kaori Kusubata ◽

Yoshihiko Tashiro ◽

Ismael Gritli ◽

Aki Sato ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Stem Cell Differentiation ◽

Cell Phenotype ◽

Bone Marrow Niche ◽

Matrix Remodeling ◽

Hematopoietic Stem

Abstract Abstract 3409 Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, stem cell maintenance and regeneration. Various stem cell niches have been shown to be hypoxic, thereby maintaining the stem cell phenotype, e.g. for hematopoietic stem cells (HSCs) or cancer stem cells. The bone marrow (BM) niche is a rich reservoir for tissue-specific pluripotent HSCs. Proteases, such as matrix metalloproteinases (MMPs) can modulate stem cell fate due to their proteolytic or non-proteolytic functions (abilities). We have investigated the role of membrane-type1 matrix metalloproteinase (MT1-MMP), known for its role in pericellular matrix remodeling and cell migration, in hematopoiesis. MT1-MMP is highly expressed in HSCs and stromal cells. In MT1-MMP−/− mice, release of kit ligand (KitL), stromal cell derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo) and interleukin-7 were impaired resulting in erythroid, myeloid and T and B lymphoid differentiation. Addition of exogenous rec. KitL and rec. SDF-1 restored hematopoiesis in vivo and in vitro. Further mechanistic studies revealed that MT1-MMP in a non-proteolytic manner activates the HIF-1 pathway, thereby inducing the transcription of the HIF-responsive genes KitL, SDF-1 and Epo. These results suggested MT1-MMP as a critical regulator of postnatal hematopoiesis, which as a modulator of the HIF pathway alters critical hematopoietic niche factors necessary for terminal differentiation and or migration. Thus, our results indicate that MT1-MMP as a key molecular link between hypoxia and the regulation of vital HSC niche factors. Disclosures: No relevant conflicts of interest to declare.

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Functional Influence of CXCL12 Regulating miRNAs in Mesenchymal Stem Cells (MSCs).

Blood ◽

10.1182/blood.v120.21.2350.2350 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2350-2350

Author(s):

Laleh S. Arabanian ◽

Fernando Fierro ◽

David M. Poitz ◽

Ruth H. Strasser ◽

Martin Bornhaeuser ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Luciferase Assay ◽

Human Mscs ◽

Bone Marrow Niche ◽

Cxcl12 Expression ◽

Hematopoietic Stem

Abstract Abstract 2350 CXCL12 is a chemokine known to be critical for the regulation of the interaction between hematopoietic stem cells (HSCs) and their niche in the bone marrow, e.g. mesenchymal stem cells (MSCs). MicroRNAs (miRNAs) are post-transcriptional regulators recently shown to mediate a variety of cellular processes in the bone marrow niche. However, identification of specific miRNAs and their regulatory role in the crosstalk between HSCs and MSCs are still poorly understood. From a library of 470 miRNAs, 26 miRNAs were shown to downregulate the levels of CXCL12 in the supernatant of the human MSC line SCP-1. Eight of them (miR-23, 130b, 135, 200b, 200c, 216, 222, 602) were chosen for further investigation according to their significant interaction with the 3'UTR of CXCL12 as determined by luciferase assay. Among them, miR-23a,130 and 222 were expressed in 46 human primary MSCs, whereas the other miRs show negligible expression in resting MSCs. However, we observed, that MSCs that underwent adipogenic and osteogenic differentiation showed strongly decreased CXCL12 protein values early (day 5) and at later stages (day 14). The later drop in CXCL12 expression was clearly associated with an increased expression of miR-23a and miR-200. We furthermore tested a subset of stimuli (proinflammatory cytokines, cytotoxic drugs, chemokines) for their ability to modulate the described miRNAs. Amongst them, exclusively the application of transforming growth factor ß1 (TGF-ß1), resulted in the induction of miR-23a and at the same time reduction of CXCL12. The effect was counteracted by transfection of anti-miR-23 molecules. Taken together, we have shown for the first time that CXCL12-targeting miRNAs (in particular miR23a) have a significant potential to regulate the properties of the stem cell niche. Moreover, miR-23 is implicated in the signalling pathway of TGF-ß1 in human MSCs. Disclosures: No relevant conflicts of interest to declare.

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Cytotoxic Exposure to Both Hematopoietic Stem and Progenitor Cells and the Bone Marrow Niche Cooperate to Promote Leukemogenesis in a Mouse Model of Therapy-Related Myeloid Neoplasms with a Del(5q)

Blood ◽

10.1182/blood.v126.23.357.357 ◽

2015 ◽

Vol 126 (23) ◽

pp. 357-357

Author(s):

Angela Stoddart ◽

Jianghong Wang ◽

Chunmei Hu ◽

Anthony A. Fernald ◽

Elizabeth M. Davis ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Alkylating Agent ◽

Conflicts Of Interest ◽

Bone Marrow Niche ◽

Cytotoxic Therapy ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Myeloid Neoplasms

Abstract A del(5q) is frequently noted in MDS, AML, and therapy-related myeloid neoplasms (t-MN) following alkylating agent therapy. Mutation/loss of the TP53 is gene found in 80% of t-MNs with a del(5q). Recent studies suggest that TP53 mutations are found at a low frequency in hematopoietic stem/progenitor cells (HSPCs) in adults (Xie et al., Nat Med 20:1472, 2014; Genovese et al., NEJM 371:2477, 2014; Jaiswal et al., NEJM 371:2488, 2014), and chemotherapy confers a selective growth advantage to these rare clones (Wong et al., Nature 518:552, 2015). We previously established a mouse model for t-MN with a del(5q) and showed that haploinsufficiency of two del(5q) genes, Egr1 and Apc, cooperate with loss of function of the Trp53 (p53) gene to induce myeloid neoplasms in mice. Specifically, transplantation of Egr1+/-, Apcdel/+ bone marrow (BM) cells transduced with p53 shRNAs into wild type (WT) recipients resulted in the development of a transplantable AML, characterized by a complex karyotype and genetic instability, in 17% of mice. There is growing evidence that microenvironment perturbations play a major role in the malignant process; however, the effect of cytotoxic therapy on HSPCs as well as the BM niche is not well understood. Using our mouse model of t-MN with a del(5q), we explored the effects of ENU, an alkylating agent, on both HSPCs and the BM microenvironment by exposing both donor and recipient mice to ENU (Panel 1 in the figure). In mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, exposure to ENU strikingly decreased survival (median survival: 200d vs. not reached) and increased the incidence of AML or MDS with multilineage dysplasia (73% vs. 17%). In the absence of p53 knockdown (i.e., control shRNA), mice survived longer (370d vs. 200d, P = 0.0014); however, 100% of mice developed MDS with dyserythropoiesis. None developed AML, suggesting that loss of p53 function is critical for leukemic transformation (Panel 2). Loss of both del(5q) genes, EGR1 and APC, was necessary to develop AML. Compared to mice transplanted with Egr1+/-, Apcdel/+, p53 shRNA HSPCs, mice transplanted with Egr1+/-, p53 shRNA HSPCs survived longer (369 d vs. 200 d, P = 0.0117) and only 40% of mice developed MDS with dysgranulopoiesis and/or dyserythropoiesis (Panel 3). None developed AML. Thus, severity of disease increases with loss of more than one del(5q) gene. Finally, to determine the separate effects of alklating agent therapy on HSPCs vs. the niche, we treated either the recipient or donor mice with ENU. Whereas ENU exposure to both donor and recipient resulted in a profound expansion of p53 shRNA+ cells and the development of MDS/AML in 73% of mice, ENU exposure of either donor or recipient led to only modest expansion of p53 shRNA+ cells and none of the mice developed MDS or AML. This suggests that the clonal expansion of cells with loss of multiple 5q genes and p53 is likely promoted by cytotoxic exposure to the cells themselves, as well as exposure to the surrounding niche cells. t-MN patients with a del(5q) typically present with trilineage dysplasia implicating all three hematopoietic cell lineages (erythroid, myeloid, and megakaryocytic) in the dysplastic process. Our mouse models shed light on some of the key genes on 5q, as well as the environmental exposure, that contributes to trilineage dysplasia in patients. Finally, our data suggests that t-MN is a "disease of the tissue", and the expansion of mutant HSPCs, e.g., with TP53 mutations, likely results from the combined effects of cytotoxic therapy on the hematopoietic cells themselves, as well as the BM microenvironment that supports hematopoiesis Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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An Altered Bone Marrow Vascular Niche Impacts Normal Hematopoiesis and Tilts the Balance Between Myelopoiesis and Lymphopoiesis

Blood ◽

10.1182/blood.v128.22.174.174 ◽

2016 ◽

Vol 128 (22) ◽

pp. 174-174

Author(s):

Cindy L Hochstetler ◽

Yuxin Feng ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Growth Factor ◽

Mouse Model ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Vascular Niche ◽

Progenitor Compartment

Abstract The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells

Cell and Tissue Research ◽

10.1007/s00441-015-2348-8 ◽

2016 ◽

Vol 364 (3) ◽

pp. 573-584 ◽

Cited By ~ 18

Author(s):

Patrick Wuchter ◽

Rainer Saffrich ◽

Stefan Giselbrecht ◽

Cordula Nies ◽

Hanna Lorig ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

In Vitro Model ◽

Model System ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

In Vitro Model System ◽

Vitro Model

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Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

Stem Cells International ◽

10.1155/2020/8885154 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Huihong Zeng ◽

Jiaoqi Cheng ◽

Ying Fan ◽

Yingying Luan ◽

Juan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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

Blood ◽

10.1182/blood-2010-09-308262 ◽

2011 ◽

Vol 117 (14) ◽

pp. 3737-3747 ◽

Cited By ~ 20

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.

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Mtss1 Suppresses BCR-ABL Induced Cell Migration and Is Downregulated in CML Stem Cells.

Blood ◽

10.1182/blood.v112.11.1077.1077 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1077-1077

Author(s):

Mirle Schemionek ◽

Shuchi Agrawal ◽

Martin Stehling ◽

Daniel G. Tenen ◽

Ashley Hamilton ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Migration ◽

Tumor Suppressor ◽

Transgenic Mice ◽

Cell Lines ◽

Actin Binding ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Retroviral Transduction

Abstract Migration and adhesion properties of hematopoietic stem cells (HSC) are disrupted in chronic myeloid leukemia (CML). Egression of these cells from the bone marrow is associated with cytoskeletal changes including actin remodeling. In a microarray screen of differentially regulated genes in HSC from BCR-ABL positive transgenic mice, we found downregulation of multiple genes involved in actin-associated changes of cell structure, adhesion, and migration (i.e. intersectin-1, cortactin, Mtss1, synaptopodin, and Gem GTPase). Mtss1 was further studied since it has been described to be a binding partner of Rac, which is essential for BCR-ABL mediated transformation, and a potential tumor suppressor. Using quanitative RT-PCR, Mtss1 downregulation (6-fold) was confirmed in HSC and unfractionated bone marrow and spleen cells from SCLtTA/BCR-ABL transgenic mice after 3 weeks of BCR-ABL induction as well as in human BCR-ABL positive cell lines. Treatment of BCR-ABL positive (32D/BCR-ABL, K562, KYO-1) but not BCR-ABL negative (32D, U937) cell lines with 5μ M Imatinib led to upregulation of Mtss1 mRNA (5- to 10-fold) and protein, suggesting that downregulation of Mtss1 is dependent on BCR-ABL kinase activity. Retroviral transduction of Mtss1 into 32D/BCR-ABL cells almost completely inhibited BCR-ABL induced cell motility of individual cells seeded on murine bone marrow stromal cells in time-lapse video experiments over the course of two hours. Interestingly, we found that retroviral transduction of Mtss1 into 32D/BCR-ABL cells completely suppressed migration of these cells to extra-hematopoietic sites in vivo upon intravenous transplantation into syngeneic C3H mice. Moreover, when Mtss1-transduced cells were injected subcutaneously, the size of the tumors was significantly decreased as compared to empty vector-transduced 32D/BCR-ABL cells (p<0.05), confirming that Mtss1 may be a tumor suppressor. These results demonstrate that Mtss1 antagonizes BCR-ABL induced cell migration and is downregulated by BCR-ABL in CML stem cells, suggesting that downregulation of Mtss1 and other cytoskeletal adaptor proteins may be required for egression of CML stem cells from the bone marrow niche. Since the same Mtss1 protein domain is responsible for both Rac and actin binding, Mtss1 may interfere with Rac function and thereby inhibit the effects of Rac on migration and cytoskeletal dynamics of HSC.

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