Reconstructing the Human Hematopoietic Niche: Opportunities for Studying Normal and Malignant Hematopoiesis,

Abstract Abstract 3412 Interactions with the hematopoietic niche in the bone marrow (BM) microenvironment are essential for hematopoietic stem cell (HSC) self-renewal. In addition, in hematological malignancies this niche is considered to serve as a sanctuary site for leukemic stem cells during chemotherapy, and to contribute to disease relapse. Although many advances have been made in understanding how the niche regulates HSC self-renewal and confers therapy resistance, most of this knowledge is based on genetic loss- or gain-of-function murine models. Since these models do not recapitulate the human physiology, there is a need for models that more closely resemble the human niche. Here, we describe a unique humanized model, which implements a novel scaffold-based technology for generating a human bone environment in RAG2−/−gc−/−-mice. Inoculation of these mice with normal human CD34+ hematopoietic progenitor cells, isolated from umbilical cord blood, resulted in homing to the human bone environment and the generation of human hematopoietic cells of distinct lineages, but more importantly also the engraftment of CD34+ cells themselves. In a next series of experiments the supportive nature of the humanized niche was further investigated with patient-derived acute myeloid leukemia (pAML) and multiple myeloma (pMM) cells, two hematopoietic malignancies that are highly dependent on the BM microenvironment for survival and growth. Inoculation of the humanized mice with pAML cells, obtained from a poor-risk patient (M1; complex karyotype) or cells from a good risk AML patient (M4; inv(16)) revealed the ability of the reconstructed human bone environment to support outgrowth of the leukemia with the cells having a similar phenotype as those from the patient sample. Interestingly, engraftment of good risk AML samples, including inv(16), has been reported to be very difficult in the NOD/SCID-based AML xenotransplant model. The humanized model that we developed was further substantiated by the ability to support the outgrowth of pMM from 7 out of 7 patients. MM is a hematological malignancy that fails to grow in mouse tissues without extra support, e.g. fetal human bone chips. Moreover, the outgrowth of pMM in our humanized model is accompanied by an increase in osteoclast activity, indicating the presence of bone resorption, one of the most relevant clinical sequelae of MM. In addition, by gene-marking pMM cells with luciferase and using bioluminescent imaging, we were able to follow myeloma outgrowth in time. Treatment of pMM-bearing mice with identical drugs as given to the patients showed that the pMM cells growing in the humanized environment in the mice responded similar as the MM patients. Hence, our model allows, for the first time, to investigate essential interactions within the human BM microenvironment for the development of normal and malignant hematopoiesis and thus for therapy development. Disclosures: de Bruijn: Xpand Biotechnology BV: Employment. Weers:Genmab BV: Employment. Parren:Genmab BV: Employment.

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Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽

10.1182/blood-2003-05-1762 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4369-4376 ◽

Cited By ~ 87

Author(s):

James C. Mulloy ◽

Jorg Cammenga ◽

Francisco J. Berguido ◽

Kaida Wu ◽

Ping Zhou ◽

...

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Cells ◽

The Self ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

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Embryonic Regulation of the Mouse Hematopoietic Niche

The Scientific World JOURNAL ◽

10.1100/2011/598097 ◽

2011 ◽

Vol 11 ◽

pp. 1770-1780 ◽

Cited By ~ 19

Author(s):

Daisuke Sugiyama ◽

Tomoko Inoue-Yokoo ◽

Stuart T. Fraser ◽

Kasem Kulkeaw ◽

Chiyo Mizuochi ◽

...

Keyword(s):

Regenerative Medicine ◽

Extrinsic Factors ◽

Self Renewal ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Healthy Donors ◽

Intrinsic Regulation ◽

Embryonic Regulation ◽

Hematopoietic Niche ◽

Transplantation Therapy

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.

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A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

Blood ◽

10.1182/blood.v126.23.246.246 ◽

2015 ◽

Vol 126 (23) ◽

pp. 246-246

Author(s):

Eva M Fast ◽

Ellen M Durand ◽

Audrey Sporrij ◽

Leslie Ojeaburu ◽

Rebecca Maher ◽

...

Keyword(s):

Gene Expression ◽

Open Chromatin ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Equity Ownership ◽

Induced Genes

Abstract Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽

10.1182/blood.v126.23.2370.2370 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2370-2370

Author(s):

Daniel Ewerth ◽

Stefanie Kreutmair ◽

Birgit Kügelgen ◽

Dagmar Wider ◽

Julia Felthaus ◽

...

Keyword(s):

Cell Cycle ◽

Research Funding ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

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Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v128.22.3870.3870 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3870-3870 ◽

Cited By ~ 4

Author(s):

Hai Cheng ◽

Patali S. Cheruku ◽

Luigi Alvarado ◽

Ayla Cash ◽

Cynthia E. Dunbar ◽

...

Keyword(s):

Cell Signaling ◽

Signaling Pathways ◽

Research Funding ◽

Fold Increase ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Human Cd34 ◽

Inflammatory Conditions ◽

Cd34 Cells

Abstract Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag's evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. Figure Figure. Disclosures Cheng: Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

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Human CD34+ stem cells express the hiwigene, a human homologue of the Drosophila genepiwi

Blood ◽

10.1182/blood.v97.2.426 ◽

2001 ◽

Vol 97 (2) ◽

pp. 426-434 ◽

Cited By ~ 125

Author(s):

Arun K. Sharma ◽

Mary C. Nelson ◽

John E. Brandt ◽

Maija Wessman ◽

Nadim Mahmud ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Amino Acid Level ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Human Leukemia ◽

Human Homologue ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34

Abstract Hematopoietic stem cells (HSCs) are characterized by their dual abilities to undergo differentiation into multiple hematopoietic cell lineages or to undergo self-renewal. The molecular basis of these properties remains poorly understood. Recently the piwigene was found in the embryonic germline stem cells (GSCs) ofDrosophila melanogaster and has been shown to be important in GSC self-renewal. This study demonstrated that hiwi, a novel human homologue of piwi, is also present in human CD34+ hematopoietic progenitor cells but not in more differentiated cell populations. Placing CD34+ cells into culture conditions that supported differentiation and rapid exit from the stem cell compartment resulted in a loss of hiwiexpression by day 5 of a 14-day culture period. Expression of thehiwi gene was detected in many developing fetal and adult tissues. By means of 5′ RACE cloning methodology, a novel putative full-length hiwi complementary DNA was cloned from human CD34+ marrow cells. At the amino acid level, the human HIWI protein was 52% homologous to the Drosophilaprotein. The transient expression of hiwi in the human leukemia cell line KG1 resulted in a dramatic reduction in cellular proliferation. Overexpression of hiwi led to programmed cell death of KG1 cells as demonstrated by the Annexin V assay system. These studies suggest that hiwi maybe an important negative developmental regulator, which, in part, underlies the unique biologic properties associated with hematopoietic stem and progenitor cells.

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JAK2V617F mutant megakaryocytes contribute to hematopoietic aging in a murine model of myeloproliferative neoplasm

10.1101/2021.08.24.457582 ◽

2021 ◽

Author(s):

Sandy Lee ◽

Helen Wong ◽

Melissa Castiglione ◽

Malea Murphy ◽

Kenneth Kaushansky ◽

...

Keyword(s):

Murine Model ◽

Matched Control ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Self Renewal ◽

Hematopoietic Stem ◽

Rna Expression Profiling ◽

Old Mice ◽

Hallmark Feature ◽

Hematopoietic Niche

Megakaryocytes (MKs) is an important component of the hematopoietic niche. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs. Using a murine model of MPN in which the human JAK2V617F gene is expressed specifically in the MK lineage, we show that the JAK2V617F-bearing MKs promote hematopoietic stem cell (HSC) aging, manifesting as myeloid-skewed hematopoiesis with an expansion of CD41+ HSCs, a reduced engraftment and self-renewal capacity, and a reduced differentiation capacity. HSCs from 2yr old mice with JAK2V617F-bearing MKs were more proliferative and less quiescent than HSCs from age-matched control mice. Examination of the marrow hematopoietic niche reveals that the JAK2V617F-bearing MKs not only have decreased direct interactions with hematopoietic stem/progenitor cells during aging, but also suppress the vascular niche function during aging. Unbiased RNA expression profiling reveals that HSC aging has a profound effect on MK transcriptomic profiles, while targeted cytokine array shows that the JAK2V617F-bearing MKs can alter the hematopoietic niche through increased levels of pro-inflammatory and anti-angiogenic factors. Therefore, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging.

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Differentiation-Chronology Specific Function of DNMT1 and Selective Anti-Leukemia Stem-Cell Therapy

Blood ◽

10.1182/blood.v112.11.201.201 ◽

2008 ◽

Vol 112 (11) ◽

pp. 201-201 ◽

Cited By ~ 2

Author(s):

Soledad Negrotto ◽

Zhenbo Hu ◽

Kevin A Link ◽

Hien Duong ◽

Andrew E. Schade ◽

...

Keyword(s):

Stem Cells ◽

Leukemia Cell ◽

Terminal Differentiation ◽

Leukemia Cells ◽

High Dose ◽

Aberrant Methylation ◽

Transcript Profile ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34

Abstract A therapeutically crucial sub-set of leukemia cells are those that self-renew (leukemia stem-cells or LSC). An open question is whether LSC self-renewal is mediated by the same pathways or factors that self-renew normal hematopoietic stem-cells (HSC). A difference in self-renewal mechanisms could form the basis for LSC-specific therapy. Hematopoietic differentiation is dependent on lineage-determining factors such as Pu.1. Pu.1 can repress or activate genes, depending on its interaction partners. We show that Pu.1 mediated terminal differentiation involves sequential repression of genes associated with self-renewal (pro-SR) (HoxB4, Bmi-1, c-Kit) followed-by activation of genes associated with differentiation (pro-DIFF)(Mcsfr, Gmcsfr, F4/80). DNA methyl-transferase 1 (DNMT1) is an arbiter of transcription repression. DNMT1 depletion concurrent with Pu.1 introduction prevents the first step of pro-SR repression and maintains self-renewal. However, DNMT1 depletion 6 hours after Pu.1 introduction (at which point pro-SR repression by Pu.1 is complete), increases differentiation. Therefore, the phenotypic consequences of DNMT1 depletion critically depend on the differentiation chronology of the cell. We then examined the effects of a leukemia first-hit event, Runx1 disruption, on Pu.1 mediated differentiation. Runx1 knock-down allowed Pu.1 mediated pro-SR repression but prevented pro-DIFF up-regulation and terminal differentiation, resulting in persistent dysregulated proliferation (self-renewal) despite pro-SR repression. DNMT1 depletion in these abnormally self-renewing cells, with an epigenetic and transcript profile distinct from parental self-renewing cells, relieved pro-DIFF repression and restored the terminal differentiation response. Therefore, DNMT1 depletion had opposite effects in the cells containing the leukemia-associated abnormality versus parental cells. Suggesting that these findings were relevant to clinical disease, leukemic bone marrow (n=130), although consisting largely of myeloblasts, demonstrated a DNA methylome (1505 CpGs, Illumina) profile that resembled differentiated cells and not precursors, with aberrant methylation concentrated at precursor signature (pro-SR) promoters and hypomethylation at differentiation signature (pro-DIFF) promoters. To therapeutically exploit these observations, we demonstrate that the nucleoside analogue Decitabine, given at doses that deplete DNMT1 without causing DNA damage, and given frequently but intermittently to allow for cell-division, produces an ideal therapeutic profile with terminal differentiation of leukemia cells but increased self-renewal of human CD34+ HSC. This effect was seen in different models of human LSC (primary human CD34+ cells transduced with either the MLL-AF9 or RUNX1-ETO leukemia fusion genes) and in primary leukemia cell samples (n=15) containing a variety of chromosome abnormalities. This therapeutic approach increased survival in a murine xeno-graft model of aggressive human MLL-AF9 leukemia. In conclusion, leukemia and LSC self-renewal, in a number of examples covering a variety of transformation-initiating abnormalities, is programmatically distinct from HSC selfrenewal. Leukemia self-renewal is associated with an epigenetic and transcript profile that reflects part-way commitment into differentiation with aberrant gene repression preventing completion of the process. This difference could explain the opposite effects of DNMT1 depletion on HSC and leukemia self-renewal, and provides the scientific foundation for differentiation (frequent metronomic low-dose) instead of cytotoxic (infrequent high-dose) regimens of epigenetically active and clinically available agents such as Decitabine.

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Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture

Blood Advances ◽

10.1182/bloodadvances.2018019315 ◽

2019 ◽

Vol 3 (7) ◽

pp. 1011-1026 ◽

Cited By ~ 5

Author(s):

Dandan Li ◽

Grace Chiu ◽

Brea Lipe ◽

Richard A. Hopkins ◽

Jacquelyn Lillis ◽

...

Keyword(s):

Culture System ◽

Ex Vivo ◽

Cxcr4 Expression ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Megakaryocytic Differentiation ◽

Cd34 Cells ◽

Hematopoietic Niche

Abstract Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

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