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Keywords:

  • aorta-gonad-mesonephros;
  • common myeloid progenitors;
  • granulocyte/macrophage progenitors;
  • self-renewal;
  • Sry related high mobility group box 17

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45lowc-Kithigh cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45lowc-Kithigh cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and β-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

In murine hematopoiesis, pluripotent hematopoietic stem cells (HSCs), which express Sca-1, c-Kit, and lack the expression of lineage markers (Lin) reside in a small fraction of the bone marrow (BM) (Ikuta & Weissman 1992). This LinSca-1+c-Kit+ (LSK) population contains all three types of multipotent hematopoietic precursors: the long-term repopulating HSC (LT-HSC), short-term repopulating HSC (ST-HSC), and the multipotent progenitors (MPPs) (Kondo et al. 1997). According to the expression of CD34 and Fcγ receptor-II/III (FcγR), the latter of which is a progenitor marker in fetal liver hematopoiesis (Lacaud et al. 1998) and mediates a wide variety of cellular responses to complexed IgG and a marker of myeloid progenitors (Ravetch & Bolland 2001), LSK cells in the BM are separated into three populations: (i) the common myeloid progenitors (CMPs), which lie in the FcγRlowCD34+ and generate all types of myeloid lineages, (ii) the granulocyte/macrophage progenitors (GMPs), which lie in the FcγRhighCD34+ and give rise to granulocyte and macrophage lineages, and (iii) the megakaryocyte/erythroid progenitors (MEPs), which lie in the FcγRlowCD34 and give rise to megakaryocytic and erythroid lineages. The CMP is considered as the precursor of the GMP and the MEP. These progenitors have limited self-renewal capacity after transplantation (Akashi et al. 2000).

Sry related high mobility group box 17 (Sox17), which is a member of the Sox family and a DNA-binding transcription factor, plays a crucial role in sustaining the viability of the foregut endoderm and acts as a differentiation regulator in the rest of the embryonic gut (Kanai-Azuma et al. 2002). Sox17 acts as a negative modulator in canonical Wnt signaling, and its silencing is an early event during tumorigenesis and contributes to the aberrant activation of Wnt signaling in colorectal carcinoma (Zhang et al. 2008). Recently, Sox17 protein has a role in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages (Uemura et al. 2013). Moreover, overexpression of Sox17 in tumor endothelial cells (tECs) was found to promote tumor angiogenesis and vascular abnormalities, while Sox17 deletion in tECs reduced tumor angiogenesis and normalized tumor vessels, thereby inhibiting tumor growth and increased the percentage of tumor-associated Mac-1+Gr-1+ myeloid cells within tumors (Yang et al. 2013). Sox17 transcription factor was implicated in the regulation of cell survival, proliferation, and maturation of hematopoietic precursors (Serrano et al. 2010). Its expression is highly restricted within the hematopoietic system to fetal and neonatal HSCs while the ectopic expression of Sox17 in multipotent adult hematopoietic progenitors of the BM has the capacity of long-term multilineage reconstitution of lethally irradiated mice and significantly increased the myeloid cells, erythroid cells, and platelets (He et al. 2011). Overexpression of Sox17 into human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) inhibited the hematopoietic differentiation of both pre-hematopoietic progenitor cells (pre-HPCs) and HPCs and reprogrammed them into hemogenic endothelial cells (ECs) (developmentally placed between ECs and pre-HPCs) (Nakajima-Takagi et al. 2013). Moreover, cells expressing Sox17 retained hemogenic potential and gave rise to hematopoietic progenitors upon inactivation of Sox17 (Nakajima-Takagi et al. 2013).

The CD45lowc-Kithigh cells of the aorta-gonad-mesonephros (AGM) region of mouse embryo at midgestation stage had the highest hematopoietic ability and was suggested to play a role in long-term reconstitution (Nobuhisa et al. 2012). The expression of Sox17 in fetal HSCs decreases during differentiation into lineage-restricted progenitors (He et al. 2011). In the current study, the role of Sox17 is investigated as a candidate regulator of the hematopoietic progenitor cells in the midgestational AGM region. Upon primary transplantation of Sox17-transduced cells in irradiated mice the absolute number of Sox17-transduced CMPs in the BM increased than other hemogenic progenitor cells. We show that Sox17-transduction into CD45lowc-Kithigh cells maintained mainly two progenitor populations, the Sox17-CMPs and the Sox17-GMPs upon several passages when co-cultured with stromal cells. Moreover, the Sox17-GMPs and the Sox17-CMPs maintained the ability to form mixed colonies when cultured on methylcellulose media and this ability was not lost even after some passages in vitro. These results imply that sustained expression of Sox17 in AGM-derived hematopoietic stem/progenitor cells maintain the self-renewal capacity and the hematopoietic ability to produce myeloid progenitors.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Animals

C57BL/6 and ICR mice were purchased from Japan SLC. Animal experiments were performed in accordance with institutional guidelines approved by the Animal Care Committee of Tokyo Medical and Dental University.

Detection of hematopoietic progenitor cells in Sox17-transduced cells

The AGM regions of C57BL/6 and ICR mice from E10.5 were excised. Isolation of CD45lowc-Kithigh cells and production of Sox17-IRES-GFP and Mock (IRES-GFP) encoding retroviruses were performed as previously described (Nobuhisa et al. 2012). Cells were immunostained with phycoerythrin (PE)-conjugated anti-mouse CD45 (30-F11), and allophycocyanin (APC)-conjugated anti-mouse c-Kit (2B8) (eBioscience, San Diego, CA, USA). Stained cells were analyzed and sorted by FACSAria (Becton Dickinson, Lincoln, NJ, USA). Sox17-transduced ball-like cells were collected and stained with biotin-conjugated lineage markers; CD4 (GK1.5), CD8 (53-6.7), Ter119 (TER-119), B220 (RA3-6B2), Gr1 (RB6-8C5), and Mac-1 (M1/70). In addition to biotin-conjugated Sca-1 (E13-161.7) antibody, the cells were incubated for 20 min on ice. The cells were further stained with Streptavidin conjugated APC-Cy7, and a combination of monoclonal antibodies containing APC-CD34 (RAM34), PE-FcγR 16/32 (93), and PE-Cy7-c-Kit (2BB). The stained cells were sorted by FACSAria. The results of flow cytometry were analyzed with FlowJo (ver 7.6.5). For the differentiation study, cells were stained with PE-Gr-1 (RB6-8C5), PE-Cy7-Mac-1 (F4/80), or APC Mac-1 (M1/70).

Co-culture with OP9 stromal cells

Sorted Sox17-transduced GFP+ cells (5 × 102) from each population were seeded on OP9 stromal cells in α-minimal essential medium (αMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 10 ng/mL interleukin (IL)-3, 50 ng/mL stem cell factor (SCF), and 10 ng/mL thrombopoietin (TPO). After 4 days, ball-like structure cells were recovered and stained with antibodies. Sorted cells (5 × 102) were recultured on new layers of OP9 stromal cells. For the long-term culture, the process was repeated every 3 or 4 days.

Cobblestone area-forming cells

Mock- and Sox17-infected cells (5 × 102) were cultured on top of a confluent monolayer of OP9 cells in αMEM supplemented with 10% (v/v) FCS, 10 ng/mL IL-3, 50 ng/mL SCF, and 10 ng/mL TPO on 12-well plates. Three to four days after culture, the number of the ball-like cell clusters and the number of cobble-stone areas containing colonies were counted.

In vitro colony-forming assay in methylcellulose

Sorted cells were cultured in MethocultTM (M3434; Stem cell technologies, Vancouver, BC, Canada). Individual colonies were scored after 7 days of culture.

Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using ISOGEN reagent according to the manufacturer's instructions (Invitrogen). cDNA was synthesized from the total RNA using Superscript III reverse transcriptase (Invitrogen). BM cells of C57BL6 mice were recovered by flushing of bone cavities (femur and tibia) and treated with NH4Cl for 5 min. The cells were washed with 5% FCS/PBS, and stained with the antibodies mentioned above. GMPs, CMPs, and MEPs of BM cells were finally sorted. Relative mRNA expression was analyzed by qRT-PCR analysis using Power SYBR Green PCR Master Mix (Applied Bioscience). Triplicate samples were run on the 7500 real-time PCR system (Applied Bioscience) and the data was subsequently analyzed by the relative standard curve method. Values for each gene were normalized to the relative quantity of β-actin. Primer sequences; β-actin, 5′-CAGCCTGGCT GGCTACGTACA-3′ 5′-CCAGGGTGTGATGGTGGGAA-3′, Sox17, 5′-TTTATGGTGTGGGCCAAAG-3′ 5′-GCCC ATGTGCGGAGACAT-3′, EPOR, 5′-GGACACCTACTT GGTATTGG-3′ 5 ‘-GACGTTGTAGGCTGGAGTCC-3′, β-major globin, 5′-CTGACAGATGCTCTCTTGGG-3′ 5′-CACAACCCCAGAAACAGACA-3′. p45NF-E2, 5′-CA CCAAATACTC CCAGGTGATATG-3′ 5′-AACTTGCCG GTAGATGACTTTAAT-3′. GATA-2, 5′-ACCACCCGATACCCACCTAT-3′ 5′-GCCATGGCAGTCACCATGCT-3′. c-Mpl, 5′-CGGTATGCCTACCGAGGAGAGAAG-3′ 5′-CGCGGGACACATTCTTCACCCAG-3′.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Myeloid potential of Sox17 gene-transduced cells in the BM

Competitor BM cells (CD45.1) and Sox17-transduced CD45lowc-Kithigh cells of E10.5 AGM cells of C57BL6 (CD45.2) cells were transplanted to 9 Gy-irradiated C57BL6 (CD45.1) recipient mice. After 3 months of transplantation, BM cells were recovered and stained with lineage markers, Sca-1, c-Kit, and CD34 antibodies, which marks the HSCs and HPCs (Cheng et al. 1996), and also with FcγR antibody. In mice transplanted with vehicle-transduced cells, donor derived cells were hardly detected (Nobuhisa et al. 2014). Sox17-transduced cells (CD45.2) showed a higher percentage of CMPs (GFP+LinSca-1c-Kit+FcγRlowCD34+ cells; Fig. 1A). The absolute number of CMPs in Sox17-transduced cell-transplanted mice increased than that of the competitor derived BM (CD45.1) cells (Fig. 1B). In contrast, GMPs (GFP+LinSca-1c-Kit+FcγRhighCD34+ cells) and MEPs (GFP+LinSca-1c-Kit+FcγRlowCD34 cells) were rare in the Sox17-transduced cell-transplanted mice. These results suggest that Sox17-transduction increased the CMPs in transplanted mice.

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Figure 1. Aberrant increase in common myeloid progenitors (CMPs) in Sry related high mobility group box 17 (Sox17)-transduced-transplanted mouse. (A) The upper panel shows the bone marrow (BM) competitor (CD45.1+) Lin Sca-1c-Kit+ cells and BM Sox17-transduced cell (CD45.2+)-transplanted mouse. The lower panel shows the expression levels of myeloid progenitors in BM competitor (CD45.1+) and Sox17-transduced cell (CD45.2+)-transplanted mouse. (B) The absolute number of CMP, granulocyte/macrophage progenitor (GMP), and megakaryocyte/erythroid progenitors (MEPs) in Sox17-transduced (CD45.2+) cells in comparison to those of competitor BM (CD45.1+) cells (n = 4). (B) (□) Competitor BM and (■) Sox17 BM.

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Sox17 directly acts on hematopoietic progenitors to increase the self-renewal

Our recent results showed that CD45lowc-Kithigh AGM cells overexpressing Sox17 were maintained at an undifferentiated state and hematopoietic differentiation was induced by down-regulation of Sox17 expression in Sox17-transduced cells (Nobuhisa et al. 2014). To examine whether Sox17 directly acts on the emergence of CMPs when E10.5 AGM CD45lowc-Kithigh cells of ICR mice are co-cultured with OP9 stromal cells, and whether it is involved in the self-renewal of CMPs, CD45lowc-Kithigh cells from E10.5 AGM cells were infected with Sox17-IRES-GFP or IRES-GFP (Mock) encoding retroviruses (Nobuhisa et al. 2004). The Sox17-overexpression in Sox17-transduced cells was confirmed by the qRT-PCR analysis and Western blotting analysis (Fig. S1A,B). After 4 days of transfection, the gating window of the GFP+LinSca-lc-Kit+ fraction was determined and the CD34 and FcγR expression profile was examined (Fig. 2A). GFP+ cells were sorted and replated on OP9 stromal cells, and these steps were repeated three times at the maximum. Sox17-transduced cells could maintain two main populations (FcγRlow and FcγRhigh) in contrast to the Mock that couldn't maintain self-renewal (Fig. 2B,C).

image

Figure 2. Self-renewal ability of Sry related high mobility group box 17 (Sox17)-transduced cells upon co-culture with stromal cell. (A) Gating strategy to isolate Sox17-transduced progenitors. (B) The upper panel shows vehicle-infected green fluorescent protein (GFP)+LinSca-1c-Kit+ cells which have no self-renewal ability. The lower panel shows Sox17-infected GFP+LinSca1c-Kit+ cells which maintained Sox17-granulocyte/macrophage progenitors (GMPs) and Sox17-common myeloid progenitors (CMPs). (C) The percentage of GMP and CMP in vehicle- or Sox17-infected cells over subsequent three passages. (C) (■) Mock-GMP, (□) Sox17-GMP, (■) Mock-CMP and (□) Sox17-CMP.

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Sox17-transduced cells contain hematopoietic progenitors with long-term potentials in vitro

The Sox17-transduced GFP+LinSca-1c-Kit+ fraction was further divided into three subpopulations according to the expression of the FcγR and CD34; Sox17-GMPs, Sox17-CMPs, and Sox17-MEPs (Fig. 3A). In this experiment, 500 cells each from Sox17-GMP and Sox17-CMP populations and 100 cells from Sox17-MEPs were sorted and plated individually on new layers of OP9 cells and cultured in the presence of cytokines (IL-3, SCF, and TPO). Sox17-MEPs were not able to proliferate in culture after passage 1 (data not shown). The Sox17-GMP population mainly maintained the high expression of both FcγR and CD34 proteins, keeping the GMP phenotype, while Sox17-GMP also produced a small fraction of Sox17-GMP-derived CMP (Fig. 3A). The Sox17-CMPs also reproduced Sox17-CMPs, but generated Sox17-GMP population as well, which was observed in all the five passages to a different extent (Fig. 3A). As shown in Figure 3B, Sox17-GMPs and Sox17-CMPs kept the formation of ball-like structures which resemble the hematopoietic cell clusters in the dorsal aorta, contain undifferentiated hematopoietic cells with self-renewal capacity, and are able to reconstitute the hematopoietic system in vivo (Nobuhisa et al. 2014). In Sox17-GMP and Sox17-CMP cultures, some differentiated colonies were produced (Fig. 3B). We suggest that Sox17-GMPs and Sox17-CMPs could maintain the self-renewal ability for at least five passages when co-cultured with OP9 cells.

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Figure 3. Self-renewal ability of myeloid progenitors in Sry related high mobility group box 17 (Sox17)-transduced aorta-gonad-mesonephros (AGM) cells. (A) Five hundred cells of Sox17-granulocyte/macrophage progenitors (GMPs) and Sox17-common myeloid progenitors (CMPs) and 100 cells of Sox17-megakaryocyte/erythroid progenitors (MEPs) were sorted and co-cultured with fresh layers of OP9 cells at each passage. Sox17-GMPs and Sox17-CMPs maintained the self-renewal ability over subsequent five passages. Box areas in flow cytometry patterns show the GMP in Sox17-GMPs and the CMP in Sox17-CMPs, respectively. Lower bar graphs show the percentage of Sox17-GMP and Sox17-CMP derived cells over the subsequent five passages. (B) Sox17-GMPs and Sox17-CMPs maintained the formation of ball-like structures (passage 2) and flattened hematopoietic colonies. (C) Five hundred cells from Sox17-GMPs and Sox17-CMPs were re-plated on freshly prepared OP9 cells with each passage, and the number of GFP+ ball-like cells was counted (n = 4). (D) Five hundred cells from Sox17-GMPs and Sox17-CMPs were re-plated on fresh OP9 cells with each passage. The number of colonies containing cobblestone-like areas was counted (n = 3). (A) (■) Sox17-GMP derived GMP, (□) Sox17-GMP derived CMP, (■) Sox17-CMP derived GMP and (□) Sox17-CMP derived CMP; (C) (■) Sox17-GMPs and (□) Sox17-CMPs; (D) (■) Sox17-GMPs and (□) Sox17-CMPs.

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Next we examined the developmental capacity of each Sox17-progenitor population. After 4 days of culture, 500 cells from Sox17-CMPs and Sox17-GMPs were sorted and plated on fresh layers of OP9 cells. These steps were repeated for five times for Sox17-CMPs and Sox17-GMPs. In each passage of each progenitor population, we counted the number of ball-like structures and cobble-stone forming areas which contain primitive hematopoietic stem cells (Nadin et al. 2003). There were no significant differences in the capability of the Sox17-CMPs and Sox17-GMPs to form the ball-like structures along the five passages (Fig. 3C). The number of the cobble-stone forming areas was slightly larger in Sox17-GMP cultures than in the Sox17-CMP counterparts (Fig. 3D). These results suggest that Sox17-CMPs and Sox17-GMPs maintain the undifferentiated state and are able to self-renew.

Differential gene expression of transcription factors of Sox17-GMPs and Sox17-CMPs

We identified genes that promote hematopoietic progenitor development from Sox17-transduced AGM cells. Quantitative RT-PCR analysis of hematopoietic genes were performed in Sox17-CMPs and Sox17-GMPs of passage 3 and also BM-CMPs, BM-GMPs, and BM-MEPs from C57BL6 mice (Fig. 4A), as Mock-GMP and Mock-CMP cells could not proliferate in culture after passage 2 and passage 1, respectively (Fig. 2B,C). Sox17 gene expression showed no discernible alteration in both Sox17-GMPs and Sox17-CMPs (Fig. 4B). We next examined the expression of c-Mpl and GATA-2. The c-Mpl gene is expressed at a higher level in CD34+HSCs/HPCs (Terstappen et al. 1991), and decreases significantly as the HSCs/HPCs differentiation occurs (Ninos et al. 2006). Akashi et al. (2000) showed that c-Mpl was expressed in the CMPs, and this population was considered the most primitive in the hematopoietic hierarchy compared with the GMPs and MEPs. CMPs commitment to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events (Akashi et al. 2000). The expression level of the c-Mpl gene in Sox17-GMPs and Sox17-CMPs was increased than that in BM-GMPs and BM-CMPs, respectively (Fig. 4B). GATA-2 is a zinc finger transcription factor which regulates the cell fate decisions of both adult and developing stem cells (Ling et al. 2004). Interestingly, the GATA-2 expression level in Sox17-GMPs was higher than that in BM-GMPs (Fig. 4B), taking the notion that GATA-2 plays a role in the proliferation and survival of hematopoietic progenitors (Kitajima et al. 2006), this may explain the proliferative ability of Sox17-GMPs in vitro culture. Nuclear factor erythroid 2 (NF-E2) is expressed in hematopoietic cells of the erythroid and megakaryocytic cell lineages (Andrews et al. 1993). In our system p45NF-E2 subunit was highly expressed in Sox17-CMPs, in comparison to its expression in Sox17-GMPs (Fig. 4B). Expression of β-globin, which is an important gene in the development of erythroid cells (Tewari et al. 1996), in Sox17-CMPs was almost double that of BM-CMPs. It was noted that ectopic Sox17-expression increased the levels of myeloid, erythroid cells and platelets (He et al. 2011). To further understand the characters of the two Sox17 populations, we detected EPOR gene, which is a specific cell surface receptor of erythropoietin (Heberlein et al. 1992). The EPOR genes expression in Sox17-GMPs and Sox17-CMPs showed approximately similar expression patterns as their BM counterparts (Fig. 4B). We conclude that upon Sox17-transduction the expression of genes involved in myelopoiesis, erythropoiesis and megakaryopoiesis are increased at least in Sox17-CMPs.

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Figure 4. Characterization of Sry related high mobility group box 17 (Sox17)-granulocyte/macrophage progenitors (GMPs) and Sox17-common myeloid progenitors (CMPs). (A) Sox17-GMPs and Sox17-CMPs (passage 3) were recovered and bone marrow (BM)-GMPs, CMPs, and megakaryocyte/erythroid progenitors (MEPs) of C57BL6 were used as a control. (B) qRT-PCR of Sox17-GMPs and Sox17-CMPs was compared with those of CMPs, GMPs, and MEPs of C57BL6 BM. (C) Sox17-transduced non-adherent cells (containing the ball-like structures and the differentiated cells) were recovered and stained with Gr-1 and Mac-1 antibodies. Percentage of Mac-1+Gr-1, Mac-1+Gr-1+, and Mac-1Gr-1+ cells in Mock-GMPs, Mock-CMPs after passage 1 and in Sox17-CMPs and Sox17-GMPs after passage 1, passage 3, and passage 5, respectively (n = 3). (D) 4 days after Sox17-transduction, Sox17-GMPs were sorted, cytospinned, and finally stained with May-Grünwald-Giemsa. Most of the cells were the early stages of myelocytic and monocytic series. (B) (■) Sox17-GMPs, (□) Sox17-CMPs, (image_n/dgd12147-gra-0001.png) BM-GMPs, (image_n/dgd12147-gra-0002.png) BM-CMPs and (image_n/dgd12147-gra-0003.png) BM-MEPs; (C) (■) Mock-GMPs, (□) Mock-CMPs, (■) Sox17-GMPs and (□) Sox17-CMPs.

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To further investigate whether Sox17-CMPs and Sox17-GMPs contain primitive cell subsets, we examined the ability of Sox17-GMPs and Sox17-CMPs to differentiate into committed cells. After passage 1, passage 3, and passage 5, we collected all the nonadherent cells in the culture from Sox17-GMPs or Sox17-CMPs, and stained the cells with Gr-1, a myeloid differentiation antigen, and Mac-1, a myeloid, monocytic and granulocytic marker and also considered one of the markers of fetal HSC (Morrison et al. 1997). In the vehicle-transduced cells, Mac-1+Gr-1+ myeloid cells represented about 50% of the Mock-GMP and Mock-CMP cultures (excluding adherent cells) after 4 days (Fig. 4C). Mac-1+Gr-1 cells represented more than 35% of the Sox17-GMPs and Sox17-CMP cultures (excluding adherent cells) after 4 days (Fig. 4C). After passage 3, this percentage was decreased in the culture (Fig. 4C; the right panel showing the data after passage 5). These two populations showed decreased number of Mac-1+Gr-1+ myeloid cells in all passages (Fig. 4C). These results showed the undifferentiated state of Sox17-transduced cells in comparison to that of the vehicle-transduced cells.

Sox17-transduced CD45lowc-Kithigh cells were cultured for 4 days and then Sox17-CMPs and Sox17-GMPs were sorted, cytospinned, and stained with May-Grünwald-Giemsa. Morphological examination revealed that Sox17-transduced cells were the early stages of myelocytic and monocytic series, respectively (Fig. 4D I and II). The Sox17-CMP and Sox17-GMP populations included cells with ring-shaped nuclei or polymorphonuclear like (PMN-like) ring cells (Fig. 4D III), which were known to include mature cells of the granulocytic lineage, monocytes, and also precursor cells of both the granulocytic and monocytic lineage (Boyce et al. 1995). These results suggest that Sox17-transduced progenitors included precursor forms of granulocytic and monocytic cells.

Sox17-GMPs and Sox17-CMPs keep the ability to form Mix-colonies in methylcellulose

In order to recognize the lineage-restricted progenitors produced after Sox17-transduction in colony forming assay, 500 cells of Sox17-GMPs and Sox17-CMPs and 100 cells of MEPs were plated in methylcellulose. After 7 days we quantified the granulocyte (G), macrophage (M), granulocyte/macrophage (GM), erythroid (E), and Mix (G-M-GM-E) colonies (Fig. 5A). Sox17-CMPs and Sox17-GMPs (passage 1) formed various colonies including G, M, GM, E, and Mix colonies. Furthermore, we studied the morphology of Mix-colony-derived cells after staining with May-Grünwald-Giemsa. Consistent with the results described above, Mix-colonies formed from Sox17-GMPs and Sox17-CMPs (passage 1) contained myelocytes and monocytes (Fig. 5D,E) and a small number of granulocytes with both eosinophilic and basophilic granules (Fig. 5D IV,E IV).

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Figure 5. The ability of Sry related high mobility group box 17 (Sox17)-granulocyte/macrophage progenitors (GMPs), Sox17-common myeloid progenitors (CMPs), and Sox17-megakaryocyte/erythroid progenitors (MEPs) to form hematopoietic colonies in semisolid medium culture along passages. (A) 500 cells of Sox17-GMPs and Sox17-CMPs, and 100 cells from Sox17-MEPs were sorted and plated on methylcellulose. The number of colonies were scored after 7 days according to the colony types G, granulocytes; M, macrophages; G/M, granulocytes and macrophages; E, erythrocytes; and Mix containing G, M, and GM colonies (n = 3). (B) Flow cytometry patterns of Sox17-GMPs and Sox17-CMPs of passage 3 and colony forming assay of Sox17-CMPs and Sox17-GMPs of passage 3 (n = 3). (C) Flow cytometry patterns of Sox17-GMPs and Sox17-CMPs of passage 5 and colony forming assay of Sox17-CMPs and Sox17-GMPs of passage 5 (n = 3). Cell morphologies of Sox17-transduced progenitors in passage 1. (D,E) One colony from each Mix-colony from each population were picked up, spread on a slide and stained with May-Grünwald-Giemsa. (D) Morphology of the Mix-colony derived from Sox17-GMPs. I. Macrophage cells, II. Myelocytes and Monocytes, III. Megakaryocyte, IV. Hybrid cell (the arrow) and V. Erythrocyte. (E) Morphology of the Mix-colony derived from Sox17-CMPs. I. Monocytes, II. Myelocytes, III. Macrophage (the arrow), IV. Hybrid cell, and V. Reticulocyte. (A, B, C) (image_n/dgd12147-gra-0004.png) E, (image_n/dgd12147-gra-0005.png) Mix, (image_n/dgd12147-gra-0006.png) GM, (image_n/dgd12147-gra-0007.png) M and (image_n/dgd12147-gra-0008.png) G.

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The BM-CMPs are common progenitors of BM-GMPs and BM-MEPs. The BM-GMPs could differentiate in granulocytes and megakaryocytes (Akashi et al. 2000). The overexpression of Sox17 in CD45lowc-Kithigh cells caused the self-renewal of Sox17-GMPs and maintained its ability to produce the Mix-colonies. To examine the Mix-colony forming ability of Sox17-CMPs and Sox17-GMPs upon several passages on culture, progenies of Sox17-GMPs and Sox17-CMPs of passage 3 were sorted, and were seeded in methylcellulose, whereas the remaining cells were cultured for the next passage. Sox17-GMPs and Sox17-CMPs of passage 3 could produce 3 fractions FcγRhighCD34+, FcγRlowCD34+, and FcγRCD34 cells (Fig. 5B). Five hundred cells of Sox17-GMP derived GMPs, Sox17-GMP derived CMPs, Sox17-CMP derived GMPs, Sox17-CMP derived CMPs and 100 cells of Sox17-GMP derived MEPs and Sox17-CMP derived MEPs were subjected to colony forming assay (Fig. 5B). After 7 days of culture Sox17-GMP derived GMPs and Sox17-GMP derived CMPs of passage 3 maintained the formation of Mix colonies, and also Sox17-CMP derived GMPs and Sox17-CMP derived CMPs of passage 3 kept the formation of Mix colonies (Fig. 5B). We further tried to increase the passage time of cells, Sox17-GMP derived cells and Sox17-CMP derived cells of passage 5 cells were cultured in methylcellulose (Fig. 5C). Both Sox17-GMPs and Sox17-CMPs derived cells of passage 5 maintained the formation of Mix-colonies (Fig. 5C). Our data implied that Sox17-transduction in CD45lowc-Kithigh cells from the E10.5 AGM increased the self-renewal ability of Sox17-progenitors and both populations kept the multipotential capacity for subsequent five passages.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Sry related high mobility group box 17 expression in fetal HSCs declines in accordance with development and lineage restriction, for example, GMP did not detectably express the Sox17 gene (He et al. 2011). Moreover, the shutdown of the exogenous Sox17 by Cre-loxP system in Sox17-transduced cells caused the induction of hematopoietic differentiation (Nobuhisa et al. 2014). Taken together we tempted to sustain Sox17 expression in CD45lowc-Kithigh cells that contained fetal HSCs, in order to know the role of Sox17, whose expression declines relatively and rapidly in hematopoietic progenitor cells.

Upon in vivo transplantation of Sox17-transduced progenitors, Sox17-CMPs increased their absolute number in comparison to Sox17-GMPs and Sox17-MEPs. However, upon in vitro culture of Sox17-transduced progenitors, we could examine the cell proliferation, self-renewal, and differentiation abilities supported mainly by cytokines, and we showed that Sox17-transduction to CD45lowc-Kithigh cells gave rise to three populations of progenitors; Sox17-GMPs, Sox17-CMPs, and Sox17-MEPs. Sox17-GMPs and Sox17-CMPs had increased self-renewal potential, while Sox17-MEPs showed no self-renewal. Previous reports showed that CD45lowc-Kithigh cells were multipotent and had the highest hematopoietic activity in vitro culture among the cells in the AGM region (Nobuhisa et al. 2012). Here, we show that Sox17-GMPs and Sox17-CMPs had the ability to form cobble-stone areas on stromal cells and Mix colonies in methylcellulose over at least five passages. Furthermore, Sox17-GMPs and Sox17-CMPs when co-cultured with OP9 cells and passed for five passages showed decreased expression of Mac-1 and Gr-1, suggesting that Sox17-transduction inhibited hematopoietic differentiation of both cell populations. Prolonged expression of Sox17 in adult HSPCs resulted in leukemic transformation, while mice transplanted with Sox17-transduced HSPCs died due to megakaryocytic/erythroid leukemia (He et al. 2011). It would be interesting to investigate mutations which might have been accumulated due to the increased cycling of Sox17-progenitor cells. Recently, mapping of Sox17-binding sites shows Sox17 activates the transcription of key regulator genes for hematopoiesis and erythrocyte differentiation (Nakajima-Takagi et al. 2013). These findings suggest that Sox17 exerts its critical function at a stage where hematopoietic progenitors are being formed. However, further studies are needed to solve out the discrepancy between in vivo and in vitro studies.

Several models exist for the differentiation of HSCs. The prevailing hypothesis of lineage commitment agrees that after the formation of MPP, lymphoid, and myeloid progenitors are segregated (Weissman 2000). In the present study, Sox17-transduced progenitors could produce G, M, GM, and E colonies upon colony forming assay of Sox17-transduced cells from the passage 1 culture, and Sox17-GMPs and Sox17-CMPs generated similar frequencies of Mix colonies in methylcellulose. Sox17-transduced cells generated cells with hybrid granules of eosinophilic and basophilic nature. These results were confirmed by picking up one colony from each Mix colonies of Sox17-transduced progenitors cultured in methylcellulose and staining it with May-Grünwald's-Giemsa. Similar to our results, Arinobu et al. (2009) noted that basophiles occurs along with the granulocyte-monocyte but not lymphoid differentiation pathway. Based on their potentials, our data indicate that Sox17-GMPs are capable of producing GMPs and CMPs for some passages, and Sox17-CMPs can produce CMPs and GMPs along the five passages. The myeloid based theory states that the prototype of blood cells is a macrophage like cell that develops to a specialized type (Kawamoto et al. 2010). Macrophage colonies were observed in Sox17-GMPs and Sox17-CMPs (Fig. 5D I,E I). These results are also compatible with those of He et al. (2011) who noted that Sox17-overexpression in adult BM cells increased myeloid and erythroid production.

We showed that this self-renewal ability displayed by Sox17-transduced progenitors likely reflects intrinsically determined changes in their properties. Overexpression of Sox17 in CD45lowc-Kithigh cells led to the expression of c-Mpl in Sox17-GMP and Sox17-CMP population. It is interesting to note that c-Mpl gene was preferentially expressed by CMPs and not GMPs of the mouse BM (Akashi et al. 2000). NF-E2 which is recognized as a critical regulator of platelet production from megakaryocytes (Fujita et al. 2013) and β-globin gene which is expressed on erythroid cells were highly expressed in Sox17-CMPs, it would be interesting to study the effect of Sox17 overexpression on the production of megakaryocytes and erythrocyte progenitors. This gene analysis indicates that Sox17-transduction increased directly the expression of c-Mpl in the two Sox17 populations, while Sox17-transduction increased the expression of NF-E2 and β-globin genes which are mainly differentiation genes in Sox17-CMPs only, and at the same time Sox17-transduction has no effect on the expression of EPOR gene in both Sox17-GMPs and Sox17-CMPs and GATA2 genes in Sox17-CMPs. Similarly, He et al. (2011) wondered whether Sox17 is capable of fully, or partially, reprogramming adult hematopoietic progenitors to a fetal type HSCs and showed that ectopic expression of Sox17 in adult HSCs and multipotent progenitors increased the self-renewal potential and the expression of fetal HSC genes. Importantly, Sox17 expression enabled long-term multilineage reconstitution (He et al. 2011). It would be interesting to examine multilineage reconstitution of Sox17-progenitors in further studies. The result of the present study show that under the influence of a single gene, Sox17-GMPs and Sox17-CMPs acquired multipotential and self-renewal abilities and maintained these properties for some passages in vitro culture. Sox17-transduction to AGM cells could be manipulated in the future to produce hematopoietic progenies for regenerative cell medicine.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank Dr T. Nakano for the OP9 cells, Ms Y. Kishikawa and Ms H. Takagi for technical support, and Dr T. Kagawa for valuable discussions. We also thank M. Fushimi for secretarial assistance, and Ms K. Inoue for technical help. This work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan. This work was in partly supported by CREST, JST and the Takeda Science Foundation. This study was also supported by the Joint Usage/Research project of Medical Research Institute, Tokyo Medical and Dental University.

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  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
dgd12147-sup-0001-FigureS1.pdfapplication/PDF265KFig. S1. (A) qRT-PCR analyses of Sox17 expression in vehicle- and Sox17-infected cells. Triplicate samples were analyzed in qRT-PCR experiments. (B) Plat-E cells were transfected with the pMY-IRES-GFP or pMY-Flag-Sox17-IRES-GFP.
dgd12147-sup-0002-FigureS1-Legend.pdfapplication/PDF47K 

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