Embryonic Regulation of the Mouse Hematopoietic Niche

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called “niche cells” surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells

Intra-Aortic Clusters Undergo Endothelial to Hematopoietic Phenotypic Transition during Early Embryogenesis

Intra-aortic clusters (IACs) attach to floor of large arteries and are considered to have recently acquired hematopoietic stem cell (HSC)-potential in vertebrate early mid-gestation embryos. The formation and function of IACs is poorly understood. To address this issue, IACs were characterized by immunohistochemistry and flow cytometry in mouse embryos. Immunohistochemical analysis revealed that IACs simultaneously express the surface antigens CD31, CD34 and c-Kit. As embryos developed from 9.5 to 10.5 dpc, IACs up-regulate the hematopoietic markers CD41 and CD45 while down-regulating the endothelial surface antigen VE-cadherin/CD144, suggesting that IACs lose endothelial phenotype after 9.5 dpc. Analysis of the hematopoietic potential of IACs revealed a significant change in macrophage CFC activity from 9.5 to 10.5 dpc. To further characterize IACs, we isolated IACs based on CD45 expression. Correspondingly, the expression of hematopoietic transcription factors in the CD45(neg) fraction of IACs was significantly up-regulated. These results suggest that the transition from endothelial to hematopoietic phenotype of IACs occurs after 9.5 dpc

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

<div><p>In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of <i>Gata2</i>, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, <i>exo utero</i> transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.</p></div

Summary of <i>exo utero</i> surgical transplantation data.

<p>Summary of <i>exo utero</i> surgical transplantation data.</p

Colony forming capacity of muscle CD45(+) c-Kit(+) cells.

<p>Muscle CD45(+) c-Kit(+) cells exhibit hematopoietic activity. (A) One thousand sorted muscle CD45(+) c-Kit(+) cells at 16.5 dpc were cultured in semisolid medium containing stem cell factor (SCF), interleukin (IL)-3, IL-6 and erythropoietin (Epo). On day 14, the number of CFU-G (colony-forming units of granulocytes), CFU-M (of macrophages), CFU-GM (of granulocytes and macrophages), CFU-Mk (of megakaryocytes), and CFU-GEMM (of granulocytes, erythrocytes, monocytes and macrophages) and the total colony number were counted. Bars represent means and SD of three culture dishes. (B) Fetal muscle CD45(+) c-Kit(+) cells obtained at 16.5 dpc were co-cultured with OP9 or OP9 Delta1 lines to assess lymphoid potential. Shown are surface expression of CD19 and B220 (B lymphoid markers) on cells cultured with OP9 cells for 16 days (left panel) and surface expression of CD4 and CD8 (T lymphoid markers) on cells cultured with OP9 Delta1 cell line for 16 days (right panel). (C) Experimental design of the colony formation assay after an organ culture step. Muscle tissue at 16.5 dpc was cultured on filter paper for 72 hours and a single cell suspension was prepared. One thousand cells were cultured and evaluated as in (A). (D) Shown are the number of cells of each colony type and total number of colonies on day 14. *<i>p</i><0.01.</p

Flow cytometric analysis of hematopoietic cells in fetal muscle tissue.

<p>Single cell suspensions of fetal muscle tissue surrounding both left and right femurs at stages 14.5 to 19.5 dpc, as analyzed by flow cytometry. (A) Surface phenotypes of fetal muscle cells. Among CD45(+) cells (upper panels), surface expression of Sca-1 and c-Kit was analyzed (lower panels). (B) The number of CD45(+) c-Kit(+) Sca-1(–) cells per two femurs and per two sets of femur-surrounding muscle tissue at 14.5 to 19.5 dpc. Bars represent mean values and SD of three individual experiments. (C) The number of CD45(+) c-Kit(+) Sca-1(+) cells per two femurs and per two sets of femur-surrounding muscle tissue at 14.5 to 19.5 dpc. Bars represent mean values and SD of three individual experiments. (D) Morphology of CD45(+) c-Kit(+) Sca-1(–) cells (left) and CD45(+) c-Kit(+) Sca-1(+) cells (right) from 16.5 dpc muscle. Cells are stained with May-Grünwald Giemsa solution. Scale bar represents 10 μm for all panels. *<i>p</i><0.01.</p

Expression of hematopoietic transcription factors and proliferation-related genes in muscle hematopoietic cells.

<p>RNA was extracted from sorted 14.5 dpc and 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(–) F4/80(–) and CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, 19.5 dpc fetal BM CD45(+) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(–) and CD45(+) c-Kit(+) Sca-1(+) cells, and expression of indicated factors assessed by real-time PCR. (A) Relative expression of hematopoietic transcription factors in the CD45(+) c-Kit(+) Sca-1(–) cell population in indicated samples. Each bar represents mean value and SD of three replicates. (B) Similar analysis in the CD45(+) c-Kit(+) Sca-1(+) cell population in indicated samples. Each bar represents mean value and SD of three technical replicates. (C) Relative expression of the proliferation-related genes <i>Myc</i> and <i>Ccnd1</i> in samples noted in (A). (D) Relative expression of proliferation-related genes in samples noted in (B). (E) Confocal images of Ki-67 in 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(+) cells. Shown is Ki-67 (green) and TOTO-3 iodide (blue) staining. Scale bar represents 50 μm for all panels. (F) The proportion of Ki-67(+) cells per total TOTO-3(+) cells in 16.5 dpc FL CD45(+) c-Kit(+) Sca-1(+) cells, 16.5 dpc muscle CD45(+) c-Kit(+) Sca-1(+) F4/80(–) cells, and 3-month-old adult BM Lin(–) CD34(–) c-Kit(+) Sca-1(+) cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138621#pone.0138621.s006" target="_blank">S5 Fig</a> shows unstained Ki-67 control. *<i>p</i><0.01.</p