A Chromatin Modifier SATB1 Promotes Lymphocyte Production From Primitive Hematopoietic Stem/Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 836-836 ◽  
Author(s):  
Yusuke Satoh ◽  
Takafumi Yokota ◽  
Hirokazu Tanaka ◽  
Koichi Kokame ◽  
Toshiyuki Miyata ◽  
...  
Keyword(s):  
Immune System ◽  
Progenitor Cells ◽  
Candidate Genes ◽  
Es Cells ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Fold Reduction ◽  
B Lineage ◽  
Satb1 Expression ◽  
Microarray Analyses

Abstract Abstract 836 Although hematopoietic stem cells (HSC) are capable for reconstituting all hematopoietic lineages in serial transplantation experiments, they undergo substantial age-related changes. Indeed, many studies have suggested that the immune system qualitatively and quantitatively changes along aging, including the selective loss of lymphopoietic potential. Lymphocyte progenitors explosively expand in fetal liver, but after birth their production sifts to bone marrow (BM) and becomes stable. Thereafter, replenishment of the adaptive immune system declines with age. If key inducers priming lymphoid lineage in HSC can be identified, they would be practical for expanding lymphocytes in culture for clinical purposes. Additionally, exogenous manipulation of their expression might boost the immune systems of immuno-compromised and/or elderly people. A primary aim of the present work was to describe molecular signatures of early stages of lymphopoiesis. Our previous studies developed a method to isolate Rag1+ early lymphocyte progenitors (ELP) from the Lineage marker- Sca-1+ c-kitHi (LSK) HSC-enriched fraction using Rag1/GFP reporter mice. We exploited the method and performed microarray analyses to compare gene expression patterns between HSC and ELP. While many molecules specific for the lymphoid lineage including T cell receptor and immunoglobulin heavy chain were highly induced at the ELP stage, some lymphoid-related genes were already expressed in the HSC-enriched fraction. In addition, our search identified many signal transduction kinases of whose function in lymphopoiesis is unknown. We then sought candidate genes governing the early lymphoid program with the goal of discovering molecules involved in the HSC to lymphoid lineage transition. Chromatin modifiers particularly drew our attention because of their ability to control spatial and temporal expression of essential genes. As a result, we identified Special AT-rich Sequence Binding 1 (SATB1) whose expression was faint in HSC and markedly up-regulated in ELP. SATB1, originally identified as a protein binding to the enhancer region of the IgH gene, is recently attracting considerable attention as a global transcription organizer for various genes. Our real-time RT-PCR analyses revealed that Satb1 expression remarkably increases along the differentiation of HSC to lymphoid-primed multipotent progenitors and ELP. In contrast, it was significantly down-regulated in the Lineage marker- Sca-1- c-kitHi myeloid progenitors. Interestingly, our analyses also found approximately 2-fold reduction of Satb1 transcripts in the Rag1/GFP- LSK cells of aged BM. In functional assays, adult BM-derived Flt3-LSK cells were retrovirally transduced with SATB1 and cultured in various conditions. In stromal cell co-cultures that support the growth of B lymphocytes and myeloid cells, the SATB1 transduction gave rise to 50–300 fold more CD19+ CD45R/B220+ Mac1- B lineage cells. In stromal-free cultures containing SCF, Flt3-ligand and IL7, SATB1 transduction also strongly induced B lineage cell production. On the other hand, methylcellulose colony assays showed 2-fold reduction of CFU-GM colony formation from SATB1-transduced cells compared to Mock-transduced cells. Next we examined if the exogenous expression of SATB1 promotes B lymphocyte growth from embryonic stem (ES) cells. SATB1 overexpression effectively promoted B1-B cell growth from mouse ES cells in OP9 co-culture system. Furthermore, we found that exogenous SATB1 expression partly restored the lymphopoietic potential in aged BM HSC that are compromised in lymphopoietic activity. Finally, we performed microarray analyses comparing SATB1- and Mock-transduced Flt3- LSK cells to seek candidate genes involved in the SATB1 function. While neither Pu.1, Ikaros, E2a, nor Notch1 was affected, some lymphoid-related genes, such as Sp4, Maf and Rag1, were increased in SATB1-overexpressed HSC. In conclusion, our results indicate that SATB1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. The findings are providing important information regarding how the earliest step of lymphopoiesis is epigenetically regulated and how modulation of the process might be useful to induce or rejuvenate the immune system. Disclosures: No relevant conflicts of interest to declare.

Satb1 Determines Hematopoietic Stem Cell Differentiation Toward the Lymphoid Lineages

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 388-388
Author(s):  
Yusuke Satoh ◽  
Takafumi Yokota ◽  
Motonari Kondo ◽  
Paul W. Kincade ◽  
Taku Kouro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Es Cells ◽  
Lymphoid Cells ◽  
The Other ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Lymphoid Lineage ◽  
Other Hand ◽  
B Lineage

Abstract Abstract 388 There is accumulating evidence that combinations of transcription factors coordinately and sequentially regulate lymphopoiesis. Five transcription factors, PU.1, Ikaros, E2A, EBF, and Pax5 are known to be hierarchically involved in early steps in B-lineage differentiation. However, it remains unclear whether the initiation of lymphoid differentiation is regulated entirely by transcription factors in a hierarchical manner. A major goal of our study was to find key genes involved in specification of lymphoid fates. For this purpose, we compared gene expression profiles between hematopoietic stem cells (HSC) and early lymphoid progenitors (ELP), which were sorted from E14.5 fetal liver of Rag1/GFP knock-in heterozygous embryos. As a result, we found that the expression of Satb1, a global chromatin regulator, was significantly increased along the differentiation of HSC to ELP. To explore roles of Satb1 in early lymphoid specification, we performed transplantation experiments, injecting HSC isolated from bone marrow of 2 weeks old Satb1-null mice into wild-type (WT) mice. We observed that Satb1-null HSC could not reconstitute CD3+ T cells in lethally irradiated WT recipients. Indeed, CD3+ T lineage recoveries from Satb1-null HSC were decreased approximately 10-fold compared with WT HSC. On the other hand, we observed varied levels of reconstitution of the B lineage and no reduction in reconstitution of the myeloid lineage resulted from Satb1 ablation. These results demonstrate that expression of Satb1 in HSC is indispensable for lymphopoiesis, but not for myelopoiesis. Furthermore, our data indicate that abnormalities of lymphoid development observed in Satb1-null mice are intrinsic to Satb1-deficient HSC. Next we conducted over-expression experiments to define the role of Satb1 in lineage fate decisions of HSC. Limiting dilution assays in MS5 co-culture condition revealed 1 in 41 Satb1-transduced Flt3−Lineage−Sca1+c-Kit+ (LSK) cells produced B cells. However, only 1 in 143 control ones were lymphopoietic under these conditions. These results suggest that Satb1 affects the lineage fate of HSC and promotes their commitment to lymphoid cells. Next we examined if the exogenous expression of Satb1 promotes B lymphocyte growth from ES cells in the OP9 co-culture system. We established ES cell clones, which can be induced to express Satb1/GFP on removal of tetracycline (Tet) from the culture medium. Eight days after Tet deprivation, 22 % of GFP+ cells expressed CD45 and CD19. On the other hand, only 1% of GFP− cells expressed same cell-surface markers. A majority of the CD19+ cells in Satb1/GFP+ ES-derived cells were positive for CD11b/Mac1 and/or CD5, suggesting that B1-B lineage cells were produced. In addition, Igh rearrangement assay revealed that DH-JH recombination occurred in the Satb1/GFP+ ES-derived cells. These results indicate that Satb1 over-expression directs even ES cells to differentiate toward the lymphoid lineage. Lymphopoietic activity becomes compromised during aging. Accumulating evidence suggests that the earliest lymphoid progenitor pools proximal to HSC are deficient in aged bone marrow. It is likely that the down-regulation of genes mediating lymphoid specification and function is involved as a major cause. Satb1 has been listed in microarray panels as one of the genes that are down-regulated in aged HSC (Rossi et al., 2005). To confirm this observation, we sorted CD150+ LSK cells from bone marrow of 6-weeks or 2-years old mice and examined expressions of Satb1. As a result, the aged CD150+LSK cells showed reduced expression (about 6-fold) of Satb1 compared with these cells from young mice. We then examined whether Satb1 expression could restore the lymphopoietic activity of progenitors derived from aged mice. Satb1-transduced Rag1/GFP− LSK cells produced significantly higher percentages of B220+ cells than control cells. With respect to the recovered B-lineage cell counts, about 3-fold more B220+ Rag1/GFP+ Mac1−cells were obtained by Satb1transduction than mock transduction. These results demonstrate that Satb1 can at least partially restore the lymphopoietic activity of aged hematopoietic progenitors. In conclusion, our results indicate that Satb1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. Such activity in generating lymphoid cells may be of clinical significance and useful to overcome immuno-senescence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells

Cell Research ◽  
2021 ◽  
Author(s):  
Chen Liu ◽  
Yandong Gong ◽  
Han Zhang ◽  
Hua Yang ◽  
Yang Zeng ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Molecular Features ◽  
Lymphoid Progenitors ◽  
B Lineage

AbstractWhereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2– CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

NF-kB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species (ROS).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1694-1694
Author(s):  
Soichi Nakata ◽  
Itaru Matsumura ◽  
Hirokazu Tanaka ◽  
Yusuke Satoh ◽  
Takumi Era ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Growth And Survival ◽  
Ros Accumulation ◽  
Dependent Growth ◽  
Induced Apoptosis

Abstract NF-kB family proteins have been reported to induce the expression of over 150 target genes, thereby crucially regulating immune responses, stress responses, and inflammation. These proteins also play important roles in cell growth and survival in various cell types. However, the precise roles of NF-kB in hematopoiesis and their mechanisms remain undetermined. To examine the roles for NF-kB family proteins in the growth and survival of hematopoietic cells, we expressed dominant negative NF-kB (IkBSR) in a murine IL-3-dependent cell line Ba/F3 using a Lac-inducible system, in which IkBSR was inducibly expressed by the IPTG treatment; this clone was designated Ba/F3/IkBSR. Furthermore, we introduced EPO receptor (R), TPOR, and G-CSFR/gp130 consisting of the extracelluar domain of G-CSFR and cytoplasmic domain of gp130 into this clone. At first, we confirmed that these clones could survive and proliferate under the cultures with IL-3, EPO, TPO, G-CSF, respectively. Although IPTG-induced IkBSR slightly suppressed IL-3- and EPO-dependent growth at low concentrations, it did not affect TPO- or gp130L-dependent growth, suggesting that NF-kB might not be so important for cytokine-dependent growth of hematopoietic cells. In contrast, IkBSR prominently enhanced factor-deprived apoptosis, which was accompanied by the ROS accumulation. To access the roles of ROS in IkBSR-enhanced apoptosis, we overexpressed ROS scavenger enzymes MnSOD and thioredoxin X (TRX) in Ba/F3/IkBSR, respectively. As a result, MnSOD and TRX significantly canceled IkB-SR-enhanced apoptosis, suggesting that ROS would be responsible for this apoptosis. We next analyzed the effects of IkBSR on the growth and survival of normal hematopoietic cells. When IkBSR was introduced into murine Lin−Sca-1+ hematopoietic stem/progenitor cells with the retrovirus system, it induced apoptosis even in the presence of appropriate cytokines. This apoptosis was also accompanied by the ROS accumulation due to the downregulated expression of anti-oxidants such as glutathione, MnSOD, glutathione peroxidase, and TRX. In addition, the expression of antiapoptotic BCl-2 family members, Bcl-XL, Bcl-2, and A1 was found to be repressed by IkBSR. However, since antioxidants such as MCI (3-methyl-1-phenyl-2-pyrazolin-5-one), N-acetylecysteine and TRX cancelled this apoptosis, ROS were supposed to be more important for IkBSR-induced apoptosis in normal hematopoietic stem/progenitor cells. To further analyze the roles for NF-kB proteins in the development of hematopoietic cells, we expressed IkBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from ES cells. When IkBSR was expressed at the stage of hemangioblasts, IkBSR induced apoptosis and inhibited the development of hematopoietic stem cells, which was also cancelled by MCI. Furthermore, when IkBSR was expressed after the development of hematopoietic stem cells, it also inhibited terminal differentiation towards granulocytes, erythrocytes, and megakaryocytes through ROS-mediated apoptosis; IkBSR inhibited granulopoiesis before the development of myeloblasts, erythropoiesis after the development of proerythroblasts, and megakaryopoiesis during polyploidization of megakaryocytes. These results indicate that NF-kB family proteins play essential roles to prevent apoptosis at multiple steps of hematopoiesis by eliminating ROS.

Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4168-4168
Author(s):  
Satiro N. De Oliveira ◽  
Francesca Giannoni ◽  
Cinnamon Hardee ◽  
Arineh Sahaghian ◽  
Laurence J N Cooper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Nsg Mice ◽  
Novel Approach ◽  
B Lineage

Abstract Abstract 4168 Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Importance of Matrix-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool

2019 ◽  
Author(s):  
P. Zhang ◽  
C. Zhang ◽  
J. Han ◽  
J. Gao ◽  
W. Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Factors Affecting ◽  
Lymphoid Lineage ◽  
3D Matrix

AbstractIn bone marrow, hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor cells (HPCs) cooperate to differentiate and replenish blood and immune cells. It has long been recognized bone marrow niche parameters interact with hematopoietic stem and progenitor cells (HSPCs) and additional work is required to study niche physical signals controlling cell behavior. Here we presented that important biophysical signals, stiffness and dimensionality, regulating expansion of bone marrow HSPCs. Mice bone marrow derived progenitor cells were cultured in collagen I hydrogel in vitro. We found stiffer 3D matrix promoted the expansion of lineage negative (Lin−) progenitor cells and Lin−Sca-1+c-kit+ (LSK) HSPCs compared to softer hydrogel. Compared with cells cultured in 2D environment, 3D embedded construct had significant advantage on HSPCs expansion, accompanied by increases on myeloid and lymphoid lineage fractions. Bright changes on gene expression were subsequently discovered. According to these data, we concluded that culture matrix dimensionality is an important factor to regulate the behavior of subpopulations in hematopoietic cell pool, which should be considered in attempts to illuminate HSCs fate decision in vitro.Statement of SignificanceWe would like to submit the enclosed manuscript entitled "Importance of Niche-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool", which we wish to be considered for publication in Biophysical Journal. Studies about the interaction between HSCs and factors provided by their microenvironment is largely focus on pure perspective of biology. But biophysical factors affecting HSC fate and behavior needs to be further explore. Herein we found ex vivo culture dimensionality affected HSPC expansion. Cell surface marker detection and mRNA expression analysis predicted the changes on myeloid and lymphoid lineage fractions. We hope niche physical signals which we identified will be considered to design HSC biomimetic niches in clinical applications. And we believe that our study will make it interesting to general readers. We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers.

scholarly journals SATB1 Expression Helps in Identification of the Lymphoid-Lineage-Biased Trajectory of Functionally Fluctuating Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 424-424
Author(s):  
Yukiko Doi ◽  
Takafumi Yokota ◽  
Yusuke Satoh ◽  
Tomoaki Ueda ◽  
Yasuhiro Shingai ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Research Funding ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Lineage Differentiation ◽  
Reporter Mice ◽  
Satb1 Expression ◽  
Serial Transplantation

Abstract Hematopoietic stem cells (HSCs) are purified well using a combination of surface markers. However, even highly enriched HSC fractions have heterogeneity in their self-renewal and differentiation potential. These seemingly contradictory roles are well regulated according to the changing demand for blood cells. Hence, interactions among lineage-related genes need to be set up, as their differentiation potential is restricted. However, how the functional diversity of HSCs reflects their intrinsic gene expression is not yet known. We previously identified special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, as a lymphoid-inducing gene in HSCs (Immunity 2013). SATB1 overexpression strongly enhanced lymphopoiesis from murine HSCs, whereas SATB1 deficiency caused HSC malfunctions. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent and differentiated preferentially to myeloid-erythroid lineages (Nat Immunol 2013). These results suggested that SATB1 is indispensable not only for the lymphopoietic potential but also for the normal function of HSCs. In this study, we first prepared hematological-lineage restricted SATB1 conditional knock out (cKO) mice to examine whether SATB1 is essential for normal HSC function in the adult bone marrow (BM). We crossed SATB1-flox mice with Cre-recombinase expressing mice under control of the Tie2 gene promoter, which efficiently inactivated the target gene in HSCs. Analyzing the BM in these mice, we observed a significant decrease in the number of HSCs as compared to those in their wild type (WT) littermates. Next, we collected HSCs from WT and Tie2-Cre SATB1-flox cKO mice using flow cytometry, and transplanted these CD45.2+ HSCs into CD45.1+ congenic mice. The chimerism of the transplanted cells was lower in recipients of SATB1 cKO mice-derived HSCs. Evaluation of the lymphocytic potential in a co-culture with MS5 stromal cells revealed that the output of lymphocytes from SATB1-cKO HSCs was lower than that of WT HSCs. Secondly, we generated SATB1 reporter mice in which SATB1 expression can be precisely monitored in vivo, and examined the early differentiation of HSCs. We found that the HSC fraction of adult BM consists of SATB1− and SATB1+ cells. We sorted the two types of HSCs with high purity and compared their growth and differentiation potential in vitro and in vivo. In methylcellulose colony assays, SATB1+ HSCs were less potent for producing myeloid-erythroid lineage colonies. In the co-culture with MS5 stromal cells, the output of lymphocytes from SATB1+ HSCs was more robust than that from SATB1− HSCs. RNA-sequencing data showed that the expression of many lymphocyte-related genes was upregulated in the SATB1+ HSCs compared to that in the SATB1− HSCs; however, there were no significant differences between the expression of stem cell-related genes in the two HSC types. In serial transplantation experiments, the SATB1+ HSCs produced more lymphocytic cells and fewer myeloid cells in the first recipients. Moreover, both types of HSCs could equally reconstitute the complete HSC fraction that contained SATB1− and SATB1+ cells, and successfully reconstituted lympho-hematopoiesis in the secondary recipients. In a study with SATB1-cKO mice, we found that SATB1 is indispensable for the preservation of the HSC potential for self-renewing proliferation and lymphocyte-differentiation. These results suggest that SATB1 plays a critical role for HSC integrity. With the newly generated SATB1 reporter mice, we confirmed the heterogeneity of HSCs. While the SATB1− and SATB1+ HSCs significantly differed in lineage-differentiation potential, both showed high long-term self-renewing capacity and reciprocal reconstitution in the serial transplantation. The cell dividing flow of the two HSC fractions settled in the same trajectory in the primary recipients, and then demonstrated equal ability for self-renewal and differentiation in the secondary recipients. Thus, we successfully isolated authentic lymphoid lineage-biased HSCs using SATB1 expression levels, and our results shed light on the oscillating nature of HSCs. Therefore, we conclude that the SATB1 expression demonstrates the fluctuation of HSCs with respect to the lineage-differentiation potential, and that SATB1 probably contributes to generation of chromatin loop formation, which endows HSCs with robust lymphopoietic potential. Disclosures Doi: Yakult Honsha Co.,Ltd.: Speakers Bureau. Yokota:SHIONOGI & CO., LTD.: Research Funding. Shibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura:Chugai Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Toyama Chemical: Research Funding; Fujimotoseiyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Shionogi: Research Funding; Alexionpharma: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding.

SATB1 Expression Marks Lymphoid-Lineage-Biased Hematopoietic Stem Cells in Mouse Bone Marrow

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2356-2356 ◽  
Author(s):  
Yukiko Doi ◽  
Takafumi Yokota ◽  
Tomohiko Ishibashi ◽  
Yusuke Satoh ◽  
Michiko Ichii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cells ◽  
System Development ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
B Lineage ◽  
Satb1 Expression ◽  
And Function

Abstract Background: Lifelong hematopoiesis is maintained by cell differentiation in which signaling pathways and transcription factors coordinately induce step-wise maturation of hematopoietic stem cells (HSCs) toward downstream effector cells. In addition, the organization of chromatin structure that creates accessible sites of target genes is also essential so as to ensure temporally and spatially adequate control of internal gene expression. Murine HSCs can be isolated with high efficiency using surface molecules including lineage-related markers, c-Kit, Sca-1, Flt3 and SLAM family proteins. However, even the highly enriched HSC fraction is still heterogeneous regarding differentiation potential, and how the HSC diversity reflects the heterogeneity of intrinsic gene-expression in HSCs is as-yet-unknown. We previously identified Special AT-rich Sequence Binding protein 1 (SATB1), a global chromatin regulator, as a lymphoid-related gene in the HSC differentiation (Satoh and Yokota et al. Immunity 2013). Indeed, SATB1 overexpression strongly enhanced both T and B lymphopoietic potential of murine HSCs whereas SATB1 deficiency caused malfunctions of HSCs in the lymphopoietic activity. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent in transplanted recipients and more prone to differentiate preferentially to myeloid-erythroid lineages (Will et al. Nat Immunol 2013). These results suggested that SATB1 is likely indispensable not only for the lymphopoietic potential but also for the integrity of HSCs. Here, to better understand the mechanism how SATB1 influences homeostatic HSC functions in adult bone marrow (BM), we have developed a new mouse model in which SATB1 expression can be precisely monitored along the HSC differentiation. Methods: The Tomato gene, coding a red fluorescent protein, was knock-in to the coding region of endogenous Satb1 gene. The heterozygous SATB1/Tomato knock-in mice in which one Satb1 allele was replaced with the Tomato were used to sort HSCs in adult BM. The sorted cells were evaluated for the differentiation potential with methylcellulose colony assays and co-cultures with MS5 stromal cells. Further, the long-term reconstitution ability was evaluated by transplantation to lethally irradiated mice. To obtain transcriptome information, total RNA was isolated from SATB1/Tomato- and SATB1/Tomato+ HSCs, and then next-generation sequencing was performed. The data were analyzed with the Ingenuity Pathway Analysis software. Results: We defined Lin- Sca1+ c-KitHi (LSK) CD150+ Flt3- cells as HSCs, especially adopting FLT3- to exclude FLT3+ lymphoid-primed multipotent progenitors from our functional analyses. We found that the LSK CD150+ Flt3- fraction contains substantial number of SATB1/Tomato+ cells. While both SATB1/Tomato- and SATB1/Tomato+ HSCs produced numerous CFU-Mix and CFU-GM/G/M colonies, the latter were less potent to produce BFU-E. In co-culture with MS5 stromal cells that support B and myeloid lineages, the output of B lineage cells from SATB1+ HSCs was more robust than that of SATB1- HSCs. Upon transplantation, enhanced B-lineage engraftment was observed in the SATB1+ HSC-transplanted recipients. Interestingly, while the two types of HSCs showed obvious difference in the differentiation potential toward lymphoid or myeloid lineage, both HSCs reconstituted the LSK CD150+ Flt3- fraction that similarly contained SATB1/Tomato- and SATB1/Tomato+ cells. With the RNA-sequencing data of SATB1- and SATB1+ HSCs, biological pathway analyses revealed that the "Hematological System Development and Function" pathway was remarkably up-regulated in the SATB1+ HSCs. Among subcategories of the "Hematological System Development and Function" pathway, the "quantity of lymphocytes" pathway was increased whereas "quantity of myeloid cells" and "quantity of granulocytes" pathways were decreased. Conclusion: We have developed a new mouse system that can be used to identify and isolate viable lymphoid-biased HSCs in the most primitive hematopoietic cell fraction of adult BM. While the SATB1- and SATB1+ HSCs differ genetically and functionally, both subtypes have displayed a self-renewal activity with mutual interconversion in transplanted recipients. These findings suggest that functional heterogeneity and variability within the HSC population is, at least in part, a manifestation of SATB1 expression. Disclosures Yokota: SHIONOGI & CO., LTD.: Research Funding.

scholarly journals STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2880-2888 ◽  
Author(s):  
Hein Schepers ◽  
Djoke van Gosliga ◽  
Albertus T. J. Wierenga ◽  
Bart J. L. Eggen ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Precise Role ◽  
Term Maintenance

Abstract The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38− cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

scholarly journals Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells

Blood ◽  
2021 ◽  
Author(s):  
Pia Sommerkamp ◽  
Mari Carmen Romero-Mulero ◽  
Andreas Narr ◽  
Luisa Ladel ◽  
Lucie Sylvie Pierrette Hustin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Comprehensive Analysis ◽  
Surface Marker ◽  
Molecular Characteristics ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Stem And Progenitor Cells ◽  
Emergency Myelopoiesis

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor populations (MPP1-4) contained within the Lin- Sca-1+ c-Kit+ (LSK) compartment have previously been identified using diverse surface marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+ CD135- CD48- CD150-). Upon transplantation, MPP5 support initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. Since MPP5 are capable of generating MPP1-4, but not HSCs, they represent a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells (HSPCs), we performed RNA-seq analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell (sc) RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with the functional reconstitution activity, MPP5 are located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

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