Plerixafor Abrogates the Supportive Function of MSC for Self-Renewal of Human Hematopoietic Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3408-3408
Author(s):  
Annette Ludwig ◽  
Rainer Saffrich ◽  
Volker Eckstein ◽  
Angela Lenze ◽  
Anke Diehlmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Division ◽  
Feeder Layer ◽  
Division Rate ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Division Rate ◽  
Cd34 Cells ◽  
Supportive Function

Abstract Abstract 3408 The CXCR4-SDF1α axis plays an important role in maintaining the stemness of human hematopoietic stem cells (HSC). In the present study we established a surrogate model for the bone marrow niche by culturing HSC on a feeder-layer of human mesenchymal stromal cells (MSC) and investigated the proliferation and differentiation behaviour upon perturbation by Plerixafor. HSC (CD34+ cells) were isolated from umbilical cord blood by fluorescent activated cell sorting (FACS). MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were stained with carboxyfluorescein succinimidyl ester (CFSE) and cultured on MSC feeder-layer for 6 days. For evaluating the influence of the culture medium on MSC cultures, three different media conditions (M1-M3) were used. Medium 1 (M1) contained 2% fetal calf serum (FCS), medium 2 (M2) contained 10% FCS and medium 3 (M3) contained GMP-grade human platelet lysate (hPL). Proliferation of HSC was calculated by analyzing the distribution of the CFSE dye (measured at day 1 and day 6). Plerixafor was added in concentrations of 0.1, 1.0 and 10.0 μM. On day 6, HSCs were harvested and analyzed by flow cytometry for CD34, CD38 and CXCR4 expressions in relationship to the cell division rate. When co-cultured with MSC, the division kinetics of HSC was increased, while the proportion of CD34+ cells remained significantly higher compared to HSC without MSC. Accordingly, more CD38− HSC were found after 6 days upon co-culture with MSC. All three MSC preparations supported self-renewing proliferation of HSC, whereas MSC M1 induced the strongest effect. This underlines that co-culture with MSC has a significant supportive function for hematopoiesis. The additional exposure to Plerixafor in the co-culture system partially reversed this effect in a dose-dependent manner: division rate of HSC and the proportion of CD34+ and CD38− cells were reduced with higher concentrations of Plerixafor. The reduction of self-renewing proliferation by Plerixafor was not observed in controls consisting of HSC without MSC. Plerixafor also rendered the CXCR4 receptors undetectable on the surface of CD34+ cells for up to 6 days, most probably due to a persisting blockade of the antibody-binding site. Human HSC co-cultured with MSC showed an increased cell division rate and produced a higher proportion of CD34+/CD38− cells. Different MSC culture media were systematically analysed in this setting and subtle differences in the supportive function could be observed. The addition of Plerixafor neutralized the effects of MSC, leading to an earlier loss of “stemness” and to lineage-commitment of HSC, thus providing evidence for the role of the CXCR4/SDF1α axis in terms of supportive function of MSC for self-renewal of HSC. Disclosures: Ho: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1397-1397
Author(s):  
Nadim Mahmud ◽  
Kazumi Yoshinaga ◽  
Craig Beam ◽  
Hiroto Araki
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (>X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (< X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

scholarly journals Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells

2018 ◽  
Vol 70 (3) ◽  
pp. 433-441
Author(s):  
Fatemeh Mohammadali ◽  
Saeid Abroun ◽  
Amir Atashi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Feeder Layer ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cord Blood Cd34 ◽  
Mild Hypoxia

Cord blood (CB) is a rich source of hematopoietic stem cells (HSC). It has been used successfully to treat a variety of hematological and non-hematological disorders. Beside the advantages of CB, its main disadvantages are the limited number of stem cells available for transplantation and delayed engraftment. Identifying strategies to enhance expansion and self-renewal of HSCs can improve transplantation efficiency. The aim of this study was to examine different culture conditions on ex vivo expansion and homeobox protein Hox-B4 gene (HOXB4) expression in cord blood CD34+ stem cells. Human cord blood CD34+ HSC were cultured in serum-free medium supplemented with cytokines with and without a feeder layer in normoxia (21% O2) and mild hypoxia (5% O2). At the end of 7 days of culture, the highest number of total nucleated cells (TNC), CD34+ cells, colony forming units (CFUs) and HOXB4 mRNA were observed in a co-culture of HSC with a bone marrow mesenchymal stem cell(MSC) feeder layer at 5% O2.We concluded that the combination of bone marrow (BM)-MSC and mild hypoxia (5% O2) not only improved HSC expansion but also enhanced HOXB4 gene expression as a self-renewal marker of HSC, and better mimicked the niche microenvironment conditions.

scholarly journals Human Thrombopoietin Knockin Mice Efficiently Support Human Hematopoiesis In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 403-403
Author(s):  
Anthony Rongvaux ◽  
Tim Willinger ◽  
Hitoshi Takizawa ◽  
Chozhavendan Rathinam ◽  
Elizabeth E. Eynon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Humanized Mice ◽  
Multilineage Differentiation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 403 Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are currently broadly used to experimentally study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. More specifically, (i) human cell engraftment is only transient, not lasting for the life of recipient mice, (ii) there is an unphysiological bias towards the lymphoid lineage as well as poor differentiation of myeloid cells, and (iii) there is an important variability in the engraftment levels between different individual animals. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. Although TPO is mouse to human cross-reactive at supraphysiological levels, we speculated that species differences would lead to insufficient TPO activity on human cells in the xenogeneic environment. We thus generated RAG2−/−γc−/− mice in which we replaced the gene encoding mouse TPO by its human homologue. This led to the expression of human TPO at human physiological levels in the serum and tissues of TPO knockin mice. Homozygous humanization of TPO (TPOh/h) led to significantly increased levels of human engraftment in the bone marrow of the hosts (an approximately 2-fold increase). TPOh/h recipients also displayed a lower engraftment variability, with an at least 80% human chimerism in 75% of the mice, and engraftment levels were maintained for longer periods of time, up to 6–7 months, while they declined after 4 months in control recipient mice. Multilineage differentiation of hematopoietic cells was also improved, with an increased ratio between granulocytes versus and lymphocytes that better reflects the physiological human blood composition. Thus, TPOh/h recipient mice provide significant improvements compared to previously available models in all three limitations listed above. Importantly, we performed phenotypical and functional analyses of human hematopoietic stem and progenitor cells in TPOh/h compared to control recipients. We observed a significant increase in the fraction of human Lin−CD34+CD38loCD90+CD45RA− cells, a population previously identified as highly enriched in functional long-term HSC. Because serial transplantation is the most stringent protocol to functionally measure the self-renewal capacity of HSCs, we purified human CD34+ cells from TPOh/h and control primary recipients and transplanted them into secondary recipients. Human CD34+ cells isolated from control primary recipients had a very low capacity to serially engraft (with human CD45+ cells detected in only 2 of 11 secondary recipients). By contrast, CD34+ cells isolated from TPOh/h primary recipients had an increased capacity to efficiently engraft secondary recipients (with human CD45+ cells present in the bone marrow of 15 of 19 secondary recipients). This result indicates that the presence of human TPO in the primary recipient favored the maintenance of human cells with enhanced self-renewal capacity. In conclusion, we demonstrate here that RAG2−/−γc−/− TPO-humanized mice efficiently support a population of cells immunophenotypically and functionally enriched in hematopoietic stem and progenitor cells. This leads to enhanced engraftment levels, better maintenance of human chimerism and improved multilineage differentiation. Therefore, RAG2−/−γc−/− TPO-humanized mice represent a novel model to study human hematopoiesis in vivo. We anticipate that this model will be useful to study human hematopoietic stem cells in vivo, with applications in the fields of hematopoiesis, hematology and hematolo-oncology. Disclosures: Stevens: Regeneron Pharmaceuticals: Employment; AnaptysBio Inc: Employment.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2617-2617
Author(s):  
Fumio Arai ◽  
Kentaro Hosokawa ◽  
Yumiko Nojima ◽  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2617 Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs. On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004). In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT. These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Pleiotrophin Regulates the Retention and Self-Renewal of Hematopoietic Stem Cells in the Bone Marrow Vascular Niche

Cell Reports ◽  
2012 ◽  
Vol 2 (4) ◽  
pp. 964-975 ◽  
Author(s):  
Heather A. Himburg ◽  
Jeffrey R. Harris ◽  
Takahiro Ito ◽  
Pamela Daher ◽  
J. Lauren Russell ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Vascular Niche ◽  
Bone Marrow Vascular Niche

scholarly journals Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques

2020 ◽  
Vol 9 (6) ◽  
pp. 1670
Author(s):  
Daniela Cilloni ◽  
Jessica Petiti ◽  
Valentina Campia ◽  
Marina Podestà ◽  
Margherita Squillario ◽  
...  
Keyword(s):  
Machine Learning ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Machine Learning Techniques ◽  
Specific Gene ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.

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

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.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2460-2460 ◽  
Author(s):  
Hairui Su ◽  
Szu-Mam Liu ◽  
Chiao-Wang Sun ◽  
Mark T. Bedford ◽  
Xinyang Zhao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Arginine Methylation ◽  
Poly I:C ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Arginine Methyltransferase ◽  
Hematopoietic Stem

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stem Cells Self-Regulate Their Differentiation To Maintain the Bone Marrow Stromal System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2335-2335
Author(s):  
Iekuni Oh ◽  
Akira Miyazato ◽  
Hiroyuki Mano ◽  
Tadashi Nagai ◽  
Kazuo Muroi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Adipocyte Differentiation ◽  
Expression Profiles ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steady Level

Abstract Mesenchymal stem cells (MSCs) account for a very small population in bone marrow stroma as a non-hematopoietic component with multipotency of differentiation into adipocytes, osteocytes and chondrocytes. MSC-derived cells are known to have hematopoiesis-supporting and immunomodulatory abilities. Although clinical applications of MSCs have already been conducted for the suppression of graft versus host disease in allogeneic stem cell transplantation and for tissue regeneration, underlying mechanisms of the biological events are still obscure. Previously, we established a differentiation model of MSCs using a mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2) (Nishikawa M et al: Blood81:1184–1192, 1993). Preadipocyte (A54) and myoblast (M1601) cell lines were cloned by treatment with 5-azacytidine. A54 cells and M1601 cells can terminally differentiate into adipocytes and myotubes, respectively, under appropriate conditions, while parent 10T1/2 cells remain undifferentiated. Moreover, A54 cells show a higher ability to support hematopoiesis compared with the other cell lines. In this study, we analyzed gene expression profiles of the three cell lines by using DNA microarray and real-time PCR to investigate molecular mechanisms for maintaining immaturity of parent 10T1/2 cells. In A54 cells, 202 genes were up-regulated, including those encoding critical factors for hematopoiesis such as SCF, Angiopoietin-1, and SDF-1 as well as genes known to be involved in adipocyte differentiation such as C/EBPα, C/EBPδ and PPAR-γ genes. These data are consistent with the hematopoiesis-supporting ability of A54 cells. During adipocyte differentiation, SCF and SDF-1 expression levels decreased in A54 cells while C/EBPα expression showed a steady level. Recently, osteoblasts have been reported to play crucial roles in “niche” for self-renewal of hematopoietic stem cells. Our results also implicate that precursor cells of non-hematopoietic components may have important roles for hematopoiesis in bone marrow. Meanwhile, in parent 10T1/2 cells, 105 genes were up-regulated, including CD90, Dlk, Wnt5α and many functionally unknown genes. Although C/EBPα expression was induced in 10T1/2 cells without differentiation under the adipocyte differentiation conditions, CD90 expression decreased, Dlk showed a steady level and Wnt5α was up-regulated. Assuming that some regulatory mechanisms are needed to keep an immature state of parent 10T1/2 cells even under the differentiation-inducible conditions, we performed following experiments. First, enforced Dlk expression in A54 cells did not inhibit terminal differentiation to adipocytes under the differentiation conditions. Second, when we cultured A54 cells in the conditioned media of parent 10T1/2 cells under the differentiation-inducible conditions, adipocyte differentiation was inhibited, suggesting that 10T1/2 cells produce some soluble molecules that can inhibit adipocyte differentiation. Since Wnt family is known to be involved in the regulation of self-renewal of several stem cells, Wnt5α may be one candidate for maintenance of “stemness” of MSCs. Taken together, the data of 10T1/2 cells suggest that MSCs can self-regulate their differentiation in the bone marrow stromal system. This concept may be important to investigate the fatty change of bone marrow in aging and in aplastic anemia.

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