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• W2805829144 abstract "•SATB1 is indispensable for both self-renewal and lymphopoiesis of adult HSCs•SATB1+ HSCs have higher reconstituting and lymphopoietic potential than SATB1− HSCs•SATB1− and SATB1+ HSCs have distinct lineage potential but are interconvertible•Difference in SATB1 expression levels contributes to HSC heterogeneity Hematopoietic stem cells (HSCs) comprise a heterogeneous population exhibiting self-renewal and differentiation capabilities; however, the mechanisms involved in maintaining this heterogeneity remain unclear. Here, we show that SATB1 is involved in regulating HSC heterogeneity. Results in conditional Satb1-knockout mice revealed that SATB1 was important for the self-renewal and lymphopoiesis of adult HSCs. Additionally, HSCs from Satb1/Tomato-knockin reporter mice were classified based on SATB1/Tomato intensity, with transplantation experiments revealing stronger differentiation toward the lymphocytic lineage along with high SATB1 levels, whereas SATB1− HSCs followed the myeloid lineage in agreement with genome-wide transcription and cell culture studies. Importantly, SATB1− and SATB1+ HSC populations were interconvertible upon transplantation, with SATB1+ HSCs showing higher reconstituting and lymphopoietic potentials in primary recipients relative to SATB1− HSCs, whereas both HSCs exhibited equally efficient reconstituted lympho-hematopoiesis in secondary recipients. These results suggest that SATB1 levels regulate the maintenance of HSC multipotency, with variations contributing to HSC heterogeneity. Hematopoietic stem cells (HSCs) comprise a heterogeneous population exhibiting self-renewal and differentiation capabilities; however, the mechanisms involved in maintaining this heterogeneity remain unclear. Here, we show that SATB1 is involved in regulating HSC heterogeneity. Results in conditional Satb1-knockout mice revealed that SATB1 was important for the self-renewal and lymphopoiesis of adult HSCs. Additionally, HSCs from Satb1/Tomato-knockin reporter mice were classified based on SATB1/Tomato intensity, with transplantation experiments revealing stronger differentiation toward the lymphocytic lineage along with high SATB1 levels, whereas SATB1− HSCs followed the myeloid lineage in agreement with genome-wide transcription and cell culture studies. Importantly, SATB1− and SATB1+ HSC populations were interconvertible upon transplantation, with SATB1+ HSCs showing higher reconstituting and lymphopoietic potentials in primary recipients relative to SATB1− HSCs, whereas both HSCs exhibited equally efficient reconstituted lympho-hematopoiesis in secondary recipients. These results suggest that SATB1 levels regulate the maintenance of HSC multipotency, with variations contributing to HSC heterogeneity. Hematopoiesis is maintained by cell differentiation, during which signaling pathways and transcription factors coordinately induce stepwise maturation of hematopoietic stem cells (HSCs) toward effector cells. During this process, HSCs lose pluripotency and acquire lineage-related functions. HSCs have been rigorously studied as the most purified among tissue stem cells in humans and mice by using a combination of various surface markers; however, even highly enriched stem cells are functionally heterogeneous at a clonal level (Copley et al., 2012Copley M.R. Beer P.A. Eaves C.J. Hematopoietic stem cell heterogeneity takes center stage.Cell Stem Cell. 2012; 10: 690-697Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Indeed, recent clonal assays of HSCs in serial transplantation revealed that the HSC fraction consists of diverse long-term reconstituting cells whose differentiation potential, particularly toward the lymphoid lineage, varies substantially (Benz et al., 2012Benz C. Copley M.R. Kent D.G. Wohrer S. Cortes A. Aghaeepour N. Ma E. Mader H. Rowe K. Day C. et al.Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs.Cell Stem Cell. 2012; 10: 273-283Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, Challen et al., 2010Challen G.A. Boles N.C. Chambers S.M. Goodell M.A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.Cell Stem Cell. 2010; 6: 265-278Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, Morita et al., 2010Morita Y. Ema H. Nakauchi H. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J. Exp. Med. 2010; 207: 1173-1182Crossref PubMed Scopus (313) Google Scholar, Yamamoto et al., 2013Yamamoto R. Morita Y. Ooehara J. Hamanaka S. Onodera M. Rudolph K.L. Ema H. Nakauchi H. Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). Slow and occasional interconversion between lymphoid-potential-positive and negative HSCs has also been reported, although lineage potential can be inherited from parental HSCs (Benz et al., 2012Benz C. Copley M.R. Kent D.G. Wohrer S. Cortes A. Aghaeepour N. Ma E. Mader H. Rowe K. Day C. et al.Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs.Cell Stem Cell. 2012; 10: 273-283Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, Shimazu et al., 2012Shimazu T. Iida R. Zhang Q. Welner R.S. Medina K.L. Alberola-Lla J. Kincade P.W. CD86 is expressed on murine hematopoietic stem cells and denotes lymphopoietic potential.Blood. 2012; 119: 4889-4897Crossref PubMed Scopus (37) Google Scholar). Elucidation of molecular mechanisms that regulate the differentiation potential of HSCs is necessary to exploit the capabilities of HSCs for various applications. Self-renewal proliferation and multilineage differentiation, which are seemingly contradictory features of HSCs, are regulated by the changing demand for blood cells in vivo. Accordingly, interactions among lineage-related genes are required to mediate the differentiation potential of HSCs (Sexton and Cavalli, 2015Sexton T. Cavalli G. The role of chromosome domains in shaping the functional genome.Cell. 2015; 160: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). We previously identified a special AT-rich-sequence-binding protein 1 (SATB1), a global chromatin organizer, as a lymphoid-lineage-inducing gene in HSCs (Satoh et al., 2013Satoh Y. Yokota T. Sudo T. Kondo M. Lai A. Kincade P.W. Kouro T. Iida R. Kokame K. Miyata T. et al.The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.Immunity. 2013; 38: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We demonstrated that exogenous induction of SATB1 in murine HSCs strongly enhanced both their T and B lymphopoietic potentials, whereas SATB1 deficiency caused malfunctions in the lymphopoietic activity of HSCs. Furthermore, another report showed that HSCs derived from the fetal livers of conventional SATB1-deficient mice were less capable of reconstituting long-term hematopoiesis in adult wild-type (WT) recipients and that SATB1-deficient HSCs differentiated preferentially into myeloid-erythroid lineages (Will et al., 2013Will B. Vogler T.O. Bartholdy B. Garrett-Bakelman F. Mayer J. Barreyro L. Pandolfi A. Todorova T.I. Okoye-Okafor U.C. Stanley R.F. et al.Satb1 regulates the self-renewal of hematopoietic stem cells by promoting quiescence and repressing differentiation commitment.Nat. Immunol. 2013; 14: 437-445Crossref PubMed Scopus (76) Google Scholar). These results suggested that SATB1 was involved in both the lymphopoietic potential and stability of HSCs. However, it remains unclear whether SATB1 plays versatile roles in homeostatic HSCs in adult bone marrow (BM) and what molecular mechanisms might contribute to these effects. In this study, we generated hematological lineage-restricted Satb1-conditional-knockout (cKO) mice to examine whether SATB1 was involved in the two fundamental but contradictory functions of HSCs (i.e., self-renewal and differentiation) in adult BM. Furthermore, we developed a reporter mouse model in which Satb1 expression was monitored during HSC differentiation in vivo. This model allowed us to identify authentic lymphoid-biased self-renewing HSCs in homeostatic adult BM. Furthermore, our data suggested that at least some lymphoid-biased HSCs fluctuate in a dynamic trajectory for self-renewal and lineage commitment. Conventional SATB1-null mice have been used to study the roles of SATB1 in hematopoiesis and lymphopoiesis (Alvarez et al., 2000Alvarez J.D. Yasui D.H. Niida H. Joh T. Loh D.Y. Kohwi-Shigematsu T. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development.Genes Dev. 2000; 14: 521-535PubMed Google Scholar, Satoh et al., 2013Satoh Y. Yokota T. Sudo T. Kondo M. Lai A. Kincade P.W. Kouro T. Iida R. Kokame K. Miyata T. et al.The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.Immunity. 2013; 38: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, Will et al., 2013Will B. Vogler T.O. Bartholdy B. Garrett-Bakelman F. Mayer J. Barreyro L. Pandolfi A. Todorova T.I. Okoye-Okafor U.C. Stanley R.F. et al.Satb1 regulates the self-renewal of hematopoietic stem cells by promoting quiescence and repressing differentiation commitment.Nat. Immunol. 2013; 14: 437-445Crossref PubMed Scopus (76) Google Scholar). However, the function of SATB1 in physiological adult hematopoiesis is difficult to discern because of neonatal mortality in these mice. To determine whether SATB1 is essential for normal hematopoiesis in adult BM, we prepared Satb1-cKO mice by crossing Satb1-floxed mice (Skowronska-Krawczyk et al., 2014Skowronska-Krawczyk D. Ma Q. Schwartz M. Scully K. Li W. Liu Z. Taylor H. Tollkuhn J. Ohgi K.A. Notani D. et al.Required enhancer-matrin-3 network interactions for a homeodomain transcription program.Nature. 2014; 514: 257-261Crossref PubMed Scopus (48) Google Scholar) with Cre-recombinase-expressing mice under control of Tie2 promoter. Analysis of the BM of these mice showed that there was a ∼25% decrease in the number of HSCs bearing lineage marker− (Lin−) Sca-1+ c-KitHi (LSK) CD150+ Flt3− in Tie2-Cre+ Satb1flox/flox mice as compared with those in their Tie2-Cre+ Satb1+/+ littermates (Figure 1A). Analyses with the Vav1-Cre-expressing model also showed similar reductions in HSCs (Figure S1A). Furthermore, cell cycle analyses of Satb1-cKO HSCs indicated that they were more likely to enter the DNA-synthesis stage than control HSCs (Figure S1B). Next, we conducted in vivo transplantation experiments to examine whether SATB1 deficiency affected the long-term reconstituting capability of HSCs. We collected HSCs from Tie2-Cre+ Satb1+/+ or Tie2-Cre+ Satb1flox/flox CD45.2+ mice by flow cytometry and transplanted these CD45.2+ HSCs into lethally irradiated CD45.1+ congenic WT mice (Figure S1C). At 4 months post-transplantation, chimerism in donor cells was significantly lower in recipients of Satb1-cKO HSCs than in those of control HSCs (Figure 1B). Transplantation with HSCs obtained from Mx1-Cre Satb1-cKO mice also provided similar results, whereas WT HSCs reconstituted long-term hematopoiesis normally in Mx1-Cre Satb1-cKO recipients (Figures 1C, S1C, and S1D). Additionally, the lineage composition of BM and peripheral blood (PB) following Satb1-cKO HSC transplantation was skewed toward the myeloid lineage (Figures 1D, 1E, S1E, and S1F). We previously reported that SATB1 plays critical roles in the early differentiation of fetal and neonatal HSCs to lymphocytes (Satoh et al., 2013Satoh Y. Yokota T. Sudo T. Kondo M. Lai A. Kincade P.W. Kouro T. Iida R. Kokame K. Miyata T. et al.The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.Immunity. 2013; 38: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To determine whether SATB1 is also important for the lymphoid lineage differentiation of adult HSCs, we cultured Tie2-Cre Satb1-cKO and control HSCs. Although both HSCs effectively produced myeloid-erythroid colonies in methylcellulose medium (Figure S1G), their growth in co-cultures with stromal cells revealed that B lymphocyte production from Satb1-cKO HSCs was reduced to ∼50% that of WT HSCs (Figure 1F). Using Mx1-Cre Satb1-cKO mice, we also confirmed that Satb1-cKO HSCs are compromised in lymphopoietic activity for T and B lineages (Figures 1G and 1H). In OP9-DL1 co-cultures, SATB1 deficiency specifically damaged the transition from DN1 to DN2 (Figure 1H). Taken together, these results supported previous observations and highlighted the versatile roles of SATB1 in adult HSCs (i.e., maintenance of HSCs in the BM, reconstitution of long-term hematopoiesis, and generation of lymphoid-lineage cells). We previously found that Satb1 expression was altered dramatically during HSC differentiation or aging (Satoh et al., 2013Satoh Y. Yokota T. Sudo T. Kondo M. Lai A. Kincade P.W. Kouro T. Iida R. Kokame K. Miyata T. et al.The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.Immunity. 2013; 38: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To visualize the intensity of Satb1 expression under physiological conditions, we generated genetically modified mice in which Tomato, the gene encoding a red fluorescent protein, was stably expressed under control of the endogenous Satb1 promoter. Tomato was knocked in at the coding region of the genomic allele of Satb1 in embryonic stem cells by homogeneous recombination, replacing one Satb1 allele with Tomato (Figure 2A). Flow cytometric analysis of mononuclear cells (MNCs) from the BM of these reporter mice showed a distinct cell fraction in the FL2 channel as compared with that in their WT littermates (Figure S2A). A previous study reported that homogeneous Satb1-null mice died shortly after birth, whereas heterozygous mice grew normally without apparent abnormalities of the immune system (Alvarez et al., 2000Alvarez J.D. Yasui D.H. Niida H. Joh T. Loh D.Y. Kohwi-Shigematsu T. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development.Genes Dev. 2000; 14: 521-535PubMed Google Scholar). Similarly, our heterozygote Satb1-reporter mice were characterized by normal body size, fertility, and lympho-hematopoiesis (Figure S2B). Using these new reporter mice, we evaluated Satb1 expression in vivo at the single-cell level. We could distinguish clusters of hematopoietic stem and/or progenitor cells based on a combination of SATB1/Tomato-, c-Kit-, and Flt3-expression intensities (Figure 2B). One prevalent model for lymphopoiesis posits that effector lymphocytes differentiate from HSCs to common lymphoid progenitors (CLPs) via lymphocyte-primed multipotent progenitors (LMPPs) (Adolfsson et al., 2005Adolfsson J. Månsson R. Buza-Vidas N. Hultquist A. Liuba K. Jensen C.T. Bryder D. Yang L. Borge O.-J. Thoren L.A.M. et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.Cell. 2005; 121: 295-306Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar, Kondo et al., 1997Kondo M. Weissman I.L. Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow.Cell. 1997; 91: 661-672Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar). In the present study, SATB1 levels increased gradually as lymphoid-lineage differentiation proceeded from HSCs but remained low in granulocyte and/or macrophage-lineage-restricted progenitors (GMPs) and megakaryocyte and/or erythrocyte-lineage-restricted progenitors (MEPs) (Figure 2B). We previously found that Satb1 expression increased as HSCs differentiated into LMPPs and CLPs (Satoh et al., 2013Satoh Y. Yokota T. Sudo T. Kondo M. Lai A. Kincade P.W. Kouro T. Iida R. Kokame K. Miyata T. et al.The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.Immunity. 2013; 38: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). To confirm whether SATB1/Tomato could be a sensitive indicator of endogenous Satb1 mRNA levels, we precisely evaluated the intensity of SATB1/Tomato in HSCs and progeny cells. Notably, the fluorescence intensity of Tomato in lymphoid-lineage progenitors increased in a stepwise manner during differentiation of the lymphoid lineage, which corresponded with the changes in mRNA levels (Figures 2C and S2C). By contrast, the intensity of SATB1/Tomato decreased as cells differentiated into GMPs and MEPs (Figures 2C and S2C). To further investigate whether Satb1 expression is useful for analysis of early differentiation into the lymphoid lineage, we crossed SATB1/Tomato-reporter mice with recombination activating 1 (RAG1)-reporter mice. We previously found that Rag1 acts as an early lymphoid lineage-related gene and developed a method to separate early lymphoid progenitors (ELPs) from HSCs by monitoring RAG1/GFP expression in vivo (Igarashi et al., 2002Igarashi H. Gregory S.C. Yokota T. Sakaguchi N. Kincade P.W. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow.Immunity. 2002; 17: 117-130Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, Yokota et al., 2003Yokota T. Kouro T. Hirose J. Igarashi H. Garrett K.P. Gregory S.C. Sakaguchi N. Owen J.J. Kincade P.W. Unique properties of fetal lymphoid progenitors identified according to RAG1 gene expression.Immunity. 2003; 19: 365-375Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Indeed, RAG1+ ELPs overlap with LMPPs and BM precursors in early thymus-immigrating cells and are depleted by pregnancy or sex steroid treatment (Lai and Kondo, 2007Lai A.Y. Kondo M. Identification of a bone marrow precursor of the earliest thymocytes in adult mouse.Proc. Natl. Acad. Sci. USA. 2007; 104: 6311-6316Crossref PubMed Scopus (82) Google Scholar, Yokota et al., 2008Yokota T. Oritani K. Garrett K.P. Kouro T. Nishida M. Takahashi I. Ichii M. Satoh Y. Kincade P.W. Kanakura Y. Soluble frizzled-related protein 1 is estrogen inducible in bone marrow stromal cells and suppresses the earliest events in lymphopoiesis.J. Immunol. 2008; 181: 6061-6072Crossref PubMed Scopus (37) Google Scholar). When we examined the double reporter mice carrying alleles of SATB1/Tomato and RAG1/GFP, the LSK fraction contained several cells with both SATB1/Tomato and RAG1/GFP expression (data not shown). However, when we added the CD150+ Flt3− criterion to purify HSCs without LMPP contamination, a substantial number of SATB1/Tomato+ cells still existed in the highly enriched HSC fraction, whereas RAG1/GFP-expressing cells were completely absent (Figure S2D). This result indicated that the SATB1/Tomato-reporter model permitted evaluation of early lymphoid differentiation of HSCs. Murine HSCs are categorized as the LSK fraction with other HSC-related markers, including CD150, CD48, Flt3, CD34, endothelial cell-selective adhesion molecule (ESAM), CD86, and CD41 (Gekas and Graf, 2013Gekas C. Graf T. CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.Blood. 2013; 121: 4463-4472Crossref PubMed Scopus (209) Google Scholar, Kiel et al., 2005Kiel M.J. Yilmaz O.H. Iwashita T. Yilmaz O.H. Terhorst C. Morrison S.J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2417) Google Scholar, Wilson et al., 2008Wilson A. Laurenti E. Oser G. van der Wath R.C. Blanco-Bose W. Jaworski M. Offner S. Dunant C.F. Eshkind L. Bockamp E. et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar, Yamamoto et al., 2013Yamamoto R. Morita Y. Ooehara J. Hamanaka S. Onodera M. Rudolph K.L. Ema H. Nakauchi H. Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, Yokota et al., 2009Yokota T. Oritani K. Butz S. Kokame K. Kincade P.W. Miyata T. Vestweber D. Kanakura Y. The endothelial antigen ESAM marks primitive hematopoietic progenitors throughout life in mice.Blood. 2009; 113: 2914-2923Crossref PubMed Scopus (56) Google Scholar). Here, we utilized the SATB1/Tomato-reporter mice to subfractionate LSK CD150+ Flt3− HSCs into SATB1/Tomato− and SATB1/Tomato+ groups and compared their expression with those of other HSC-related markers. The result showed that SATB1/Tomato− HSCs had higher average levels of CD150 and ESAM and lower average levels of CD48 and CD34 than SATB1/Tomato+ HSCs (Figure 2D), suggesting that SATB1/Tomato− HSCs were likely to contain more undifferentiated cells than SATB1/Tomato+ HSCs. Additionally, the fluorescence intensity of CD86, a reported lymphoid-lineage-related marker (Shimazu et al., 2012Shimazu T. Iida R. Zhang Q. Welner R.S. Medina K.L. Alberola-Lla J. Kincade P.W. CD86 is expressed on murine hematopoietic stem cells and denotes lymphopoietic potential.Blood. 2012; 119: 4889-4897Crossref PubMed Scopus (37) Google Scholar), was higher in SATB1/Tomato+ HSCs than in SATB1/Tomato− HSCs (Figure S2E). These results indicated that the highly enriched HSC fraction was still heterogeneous and could be sorted into different categories according to SATB1/Tomato-expression levels. We then conducted in vitro experiments to evaluate the functional characteristics of SATB1/Tomato− and SATB1/Tomato+ HSCs. We sorted both fractions and confirmed high-purity sorting (Figures 3A and 3B ) and also confirmed that the fluorescence intensities of Tomato reflected Satb1-transcript levels (Figure 3C). We cultured the sorted HSCs in methylcellulose medium and evaluated the growth of myeloid and erythroid colonies. The SATB1+ HSCs showed a significantly lower capability to produce all types of colonies, including colony-forming unit (CFU)-Mix, CFU-granulocyte-monocyte (GM)/G/M, and erythroid burst-forming unit (BFU-E), than SATB1− HSCs (Figure 3D). To evaluate simultaneous differentiation into lymphoid and myeloid lineages in each fraction of HSCs, we performed co-cultures with MS5 stromal cells capable of supporting both lymphoid and myeloid growth. Under these conditions, SATB1+ HSCs gave rise to a robust population of hematopoietic cells, including myeloid cells (Figure 3E). Additionally, we observed marked differences in lymphopoietic potential between the two HSCs. After 1 week of culture, SATB1+ HSCs generated a large number of B-lymphocytes, which continued to expand, whereas SATB1− HSCs did not efficiently produce lymphocytes (Figure 3E). Given the heterogeneity of LSK CD150+ Flt3− cells, the discrepancy in myeloid-lineage growth between the two culture conditions might reflect that the CFU assays measure the clonal growth of rather committed hematopoietic progenitors, whereas MS5 co-cultures support more immature progenitors. These results demonstrated that SATB1− and SATB1+ HSCs substantially differed in the growth and differentiation potential, particularly in the lymphoid lineage. To obtain comprehensive information about gene expression in SATB1− and SATB1+ HSCs, we performed next-generation RNA sequencing (RNA-seq) analyses. The results revealed that 509 genes, including 316 upregulated and 193 downregulated genes, were significantly altered (p < 0.05) in SATB1/Tomato+ HSCs (Figure 4A), indicating that SATB1+ HSCs were transcriptionally more active than SATB1− HSCs. Among the top 10 genes that were down- or upregulated (p < 0.05), we found that the expression of several lymphoid-lineage-related genes was significantly induced in SATB1+ HSCs (Figure 4B). Biological pathway analyses using Ingenuity Pathway Analysis (IPA) software revealed that the “hematological system development and function” and “hematopoiesis” pathways were upregulated in SATB1+ HSCs (Figure S3A). Among subcategories of hematopoiesis, 18 pathways were predicted to increase, including differentiation of lymphocytes (p = 1.09E−24; Figure 4C) and development of hematopoietic progenitor cells (p = 9.08E−14; Figure 4C). Additionally, nine of 18 increased pathways were involved in lymphoid-lineage cell generation (Figure 4C). Evaluation of the total pathways among subcategories of hematological system development and function showed that lymphocyte-differentiation-related pathways were markedly increased (Figure S3B). We also tested the expression of genes related to hematopoietic lineage-fate decision in SATB1− and SATB1+ HSCs by real-time PCR. We observed that lymphoid-lineage-related genes were generally upregulated (Figure 4D), whereas myeloid-lineage-related genes and erythroid-lineage-related genes were downregulated in SATB1+ HSCs (Figure 4E). The expression of genes required for preservation of HSC stemness was not significantly altered (Figure 4F). Notably, the expression of nuclear factor of activated T cells, reportedly involved in maintaining the lymphoid-lineage-differentiating capacity in HSCs (Luchsinger et al., 2016Luchsinger L.L. de Almeida M.J. Corrigan D.J. Mumau M. Snoeck H.W. Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential.Nature. 2016; 529: 528-531Crossref PubMed Scopus (154) Google Scholar), was lower in SATB1+ HSCs. Furthermore, expression of the nuclear receptor subfamily 4 group A member 1, a marker of myeloid-biased HSCs (Land et al., 2015Land R.H. Rayne A.K. Vanderbeck A.N. Barlowe T.S. Manjunath S. Gross M. Eiger S. Klein P.S. Cunningham N.R. Huang J. et al.The orphan nuclear receptor NR4A1 specifies a distinct subpopulation of quiescent myeloid-biased long-term HSCs.Stem Cells. 2015; 33: 278-288Crossref PubMed Scopus (17) Google Scholar), was significantly decreased in SATB1+ HSCs (Figure 4F). We performed transplantation assays to evaluate the differentiation potential of SATB1− and SATB1+ HSCs in vivo. HSCs are defined functionally by their ability to serially engraft in lethally irradiated recipients and regenerate their hematopoietic and immune systems. SATB1/Tomato− and SATB1/Tomato+ HSCs were sorted from adult BM of CD45.2+ SATB1/Tomato-reporter mice, and 1,000 cells from each HSC fraction were independently transplanted into lethally irradiated CD45.1+ congenic WT mice along with 2 × 105 rescue BM cells (Figure S4A). After 4 months, we sacrificed the recipients and examined their PB and BM. We found that, although both SATB1− and SATB1+ HSCs engrafted in the BM, the latter contributed to recipient hematopoiesis with higher chimerism than the former (Figure 5A). This result was unexpected, because SATB1+ HSCs exhibited a more differentiated phenotype on average in terms of cell-surface antigens (Figure 2D). In addition to engrafting activity, the pattern of lineage reconstitution varied considerably between the two HSC types. We observed that lymphocyte output was significantly higher, whereas myeloid-cell generation was lower in SATB1+ HSC-transplanted recipients (Figure S4B). To assess whether this imbalance resulted from functional differences in the two types of HSCs in terms of lineage-restricted progenitors, we evaluated the proportions of CLPs and myeloid progenitors among the engrafted donor cells. Notably, significantly more CLPs were produced from SATB1+ HSCs than from SATB1− HSCs, whereas myeloid progenitors were more efficiently generated from the latter HSCs (Figure 5B). Although the two types of HSCs showed obvious differences in their ability to differentiate into either lymphoid or myeloid lineages, both HSCs sufficiently reconstituted the LSK CD150+ Flt3− HSC-enriched fraction, which contained a variety of HSCs in terms of SATB1/Tomato expression (Figures 5C and 5D). Therefore, SATB1− HSCs reconstituted the other HSCs exhibiting positive SATB1/Tomato intensities and vice versa. These results suggested that SATB1− and SATB1+ HSCs are mutually interconvertible and that both SATB1− and SATB1+ fractions contained the definitive characteristics of HSCs. To further examine the long-term repopulating ability of each HSC fraction, we performed serial-transplant assays. BM cells were collected from primary recipients of each group and were transplanted into lethally irradiated secondary CD45.1+ recipients (Figure S4A). After 4 months, we observed high contribution of CD45.2+ cells in both types of recipients; however, the donor-cell chimerism in the BM did not differ (Figure 5E). Additionally, both HSCs generated lymphocytes and myeloid cells in the PB, as well as in CLPs, myeloid progenitors, and HSCs, in the BM with equal efficiency (Figures 5F, 5G, and S4C). The intensity of SATB1/Tomato in the reconstituted HSCs was also similar (Figure 5H). These observations suggested that, although SATB1− and SATB1+ HSCs differed in lineage-output potential, they were interconvertible and retained multipotency as authentic HSCs over the long term. As described, SATB1+ HSCs engrafted more efficiently than SATB1− HSCs, contrary to our expectations (Figure 5A). Because the transplantations were performed with a bulk fraction containing a diverse cell population, a small population of SATB1+ cells at the threshold level might have contributed to the high engraftment. To address this issue, we segmented the LSK CD150+ Flt3− fraction into five subfractions according to SATB1/Tomato intensity and performed transplantation with only 10 cells of each subfraction in five groups of recipient mice (" @default.
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• W2805829144 date "2018-06-01" @default.
• W2805829144 modified "2023-10-15" @default.
• W2805829144 title "Variable SATB1 Levels Regulate Hematopoietic Stem Cell Heterogeneity with Distinct Lineage Fate" @default.
• W2805829144 cites W1482479272 @default.
• W2805829144 cites W1956972180 @default.
• W2805829144 cites W1964877471 @default.
• W2805829144 cites W1967662525 @default.
• W2805829144 cites W1969930387 @default.
• W2805829144 cites W1975303324 @default.
• W2805829144 cites W1978217382 @default.
• W2805829144 cites W1980148430 @default.
• W2805829144 cites W1982130774 @default.
• W2805829144 cites W1984850507 @default.
• W2805829144 cites W1992097145 @default.
• W2805829144 cites W2010289839 @default.
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• W2805829144 cites W2070154483 @default.
• W2805829144 cites W2076623492 @default.
• W2805829144 cites W2077330278 @default.
• W2805829144 cites W2093931465 @default.
• W2805829144 cites W2114514472 @default.
• W2805829144 cites W2127090737 @default.
• W2805829144 cites W2141874645 @default.
• W2805829144 cites W2152414109 @default.
• W2805829144 cites W2156475356 @default.
• W2805829144 cites W2159082795 @default.
• W2805829144 cites W2295313298 @default.
• W2805829144 cites W2602848567 @default.
• W2805829144 cites W2793006183 @default.
• W2805829144 cites W4240523393 @default.
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