scholarly journals C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 28-28
Author(s):  
Nicole D. Vincelette ◽  
Jungwon Moon ◽  
Andrew T. Kuykendall ◽  
Ling Zhang ◽  
Rami S. Komrokji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Myeloid Cell ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myeloid Progenitors

Abstract Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs. To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice vs. -MYC +/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin -Sca-1 +cKit + [LSK]), multipotent progenitors (MPPs; LSK CD48 +CD150 -), common myeloid progenitors (CMPs; Lin -Sca1 -cKit +), granulocyte-monocyte progenitors (GMPs; Lin -Sca-1 -cKit +CD34 +FCγR +), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease. We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre +/-;Rosa26-LSL-MYC +/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit + and Cd11b + BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER T2+/-;Myc fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN. Disclosures Kuykendall: Pharmaessentia: Honoraria; Abbvie: Honoraria; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Blueprint: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria, Speakers Bureau. Komrokji: Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; Geron: Honoraria; Novartis: Honoraria; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau.

Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1260-1266 ◽  
Author(s):  
Sebastian Stier ◽  
Tao Cheng ◽  
Randolf Forkert ◽  
Christoph Lutz ◽  
David M. Dombkowski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cell Cycle Checkpoints ◽  
Hematopoietic Stem ◽  
Cell Cycling

Abstract Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult–derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.

scholarly journals Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

2013 ◽  
Vol 18 (1) ◽  
Author(s):  
Viviana Rodríguez-Pardo ◽  
Jean Vernot
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Cell Subpopulation ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Secreted Factors

AbstractThe purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

scholarly journals Calreticulin haploinsufficiency augments stem cell activity and is required for onset of myeloproliferative neoplasms

Blood ◽  
2020 ◽  
Author(s):  
Kotaro Shide ◽  
Takuro Kameda ◽  
Ayako Kamiunten ◽  
Yoshinori Ozono ◽  
Yuki Tahira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Cell Activity ◽  
Extramedullary Hematopoiesis ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Deficient Mice

Mutations in JAK2, MPL, or CALR are detected in more than 80% of myeloproliferative neoplasm (MPN) patients and are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow, and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of hematopoietic stem cells. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of hematopoietic stem cells damaged by CALR mutations. Only recipient mice transplanted with Lineage－Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was required for the onset of CALR-mutated MPNs.

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.

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.

Cell staining for sorting of hematopoietic stem cells (HSC) and myeloid progenitors and isolating RNA from sorted cells

2006 ◽  
Author(s):  
Hideyo Hirai ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Christopher Hetherington ◽  
Shin-ichi Mizuno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Staining ◽  
Myeloid Progenitors

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.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

2020 ◽  
Vol 15 (3) ◽  
pp. 250-262
Author(s):  
Maryam Islami ◽  
Fatemeh Soleimanifar
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Umbilical Cord ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Future Directions ◽  
Hematopoietic Stem ◽  
Efficient Procedure ◽  
Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

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