Identification of fetal liver stromal subsets in spectral cytometry using the parameter autofluorescence

The fetal liver is the main hematopoietic organ during embryonic development. The fetal liver is also the unique anatomical site where hematopoietic stem cells expand before colonizing the bone marrow, where they ensure life-long blood cell production and become mostly resting. The identification of the different cell types that comprise the hematopoietic stroma in the fetal liver is essential to understand the signals required for the expansion and differentiation of the hematopoietic stem cells. We used a panel of monoclonal antibodies to identify fetal liver stromal cells in a 5-laser equipped spectral flow cytometry analyzer. The ″Autofluorescence Finder″ of SONY ID7000 software identified two distinct autofluorescence emission spectra. Using autofluorescence as a fluorescence parameter we could assign the two autofluorescent signals to three distinct cell types and identified surface markers that characterize these populations. We found that one autofluorescent population corresponds to hepatoblasts and cholangiocytes whereas the other expresses mesenchymal transcripts and was identified as stellate cells. Importantly, after birth, autofluorescence becomes the unique identifying property of hepatoblasts because mature cholangiocytes are no longer autofluorescent. These results show that autofluorescence used as a parameter in spectral flow cytometry is a useful tool to identify new cell subsets that are difficult to analyze in conventional flow cytometry.

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Different Roles of Glycosylphosphatidylinositol in Various Hematopoietic Cells as Revealed by a Mouse Model of Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood.v94.9.2963.421k18_2963_2970 ◽

1999 ◽

Vol 94 (9) ◽

pp. 2963-2970 ◽

Cited By ~ 2

Author(s):

Y. Murakami ◽

T. Kinoshita ◽

Y. Maeda ◽

T. Nakano ◽

H. Kosaka ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Cell Clone ◽

Early Stage ◽

Fetal Liver ◽

Cell Types ◽

Gpi Anchor ◽

Hematopoietic Stem ◽

Linked Gene

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or a few clones of mutant hematopoietic stem cells defective in glycosylphosphatidylinositol (GPI) synthesis as a result of somatic mutation in the X-linked gene PIG-A. The mutant stem cell clone dominates hematopoiesis by a mechanism that is unclear. To test whether a lack of multiple GPI-anchored proteins results in dysregulation and expansion of stem cells, we generated mice in which GPI-anchor negative cells are present only in the hematopoietic system. We transplanted lethally irradiated mice with female fetal liver cells bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages contained GPI-anchor negative cells. The percentage of GPI-anchor negative cells was much higher in T lymphocytes including immature thymocytes than in other cell types, suggesting a regulatory role for GPI-anchored proteins at an early stage of T-lymphocyte development. However, the proportions of GPI-anchor negative cells in various blood cell lineages were stable over a period of 42 weeks, indicating thatPiga mutation alone does not account for the dominance of the mutant stem cells and that other phenotypic changes are involved in pathogenesis of PNH.

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Maturation of hematopoietic stem cells from prehematopoietic stem cells is accompanied by up-regulation of PD-L1

Journal of Experimental Medicine ◽

10.1084/jem.20161594 ◽

2017 ◽

Vol 215 (2) ◽

pp. 645-659 ◽

Cited By ~ 9

Author(s):

Joanna Tober ◽

Marijke M.W. Maijenburg ◽

Yan Li ◽

Long Gao ◽

Brandon K. Hadland ◽

...

Keyword(s):

Stem Cells ◽

Immune Responses ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Programmed Death

Hematopoietic stem cells (HSCs) mature from pre-HSCs that originate in the major arteries of the embryo. To identify HSCs from in vitro sources, it will be necessary to refine markers of HSCs matured ex vivo. We purified and compared the transcriptomes of pre-HSCs, HSCs matured ex vivo, and fetal liver HSCs. We found that HSC maturation in vivo or ex vivo is accompanied by the down-regulation of genes involved in embryonic development and vasculogenesis, and up-regulation of genes involved in hematopoietic organ development, lymphoid development, and immune responses. Ex vivo matured HSCs more closely resemble fetal liver HSCs than pre-HSCs, but are not their molecular equivalents. We show that ex vivo–matured and fetal liver HSCs express programmed death ligand 1 (PD-L1). PD-L1 does not mark all pre-HSCs, but cell surface PD-L1 was present on HSCs matured ex vivo. PD-L1 signaling is not required for engraftment of embryonic HSCs. Hence, up-regulation of PD-L1 is a correlate of, but not a requirement for, HSC maturation.

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Cultivation of aorta-gonad-mesonephros–derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow

Blood ◽

10.1182/blood.v99.4.1190 ◽

2002 ◽

Vol 99 (4) ◽

pp. 1190-1196 ◽

Cited By ~ 41

Author(s):

Masaki Takeuchi ◽

Takashi Sekiguchi ◽

Takahiko Hara ◽

Taisei Kinoshita ◽

Atsushi Miyajima

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Developmental Process ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Coculture System

During mammalian development, definitive hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) region and colonize the fetal liver (FL) before hematopoiesis occurs in the bone marrow. The FL is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated along with functional maturation of hepatic cells as a metabolic organ. To characterize HSCs and FL microenvironments during development, this study establishes a coculture system composed of AGM-originated HSCs and FL nonhematopoietic cells. The results demonstrate that FL cells support significant expansion of lineage-committed hematopoietic cells as well as immature progenitors. More important, long-term repopulating activity was amplified from AGM-originated HSCs in this coculture system. Engraftment of HSCs to the bone marrow was strongly enhanced by coculture. In addition, AGM HSCs produced significantly more hematopoietic cells than E14.5 and E18.5 FL HSCs in vitro. These results suggest that the FL microenvironment not only stimulates expansion of the hematopoietic system, but also possibly modifies the characteristics of AGM HSCs. Thus, this coculture system recapitulates the developmental process of HSCs and the FL microenvironment and provides a novel means to study the development of hematopoiesis.

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Two Distinct Precursors of Hematopoietic Stem Cells and Endothelial Progenitors Characterized by the PCLP1 Expression.

Blood ◽

10.1182/blood.v106.11.2274.2274 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2274-2274

Author(s):

Izumi Onitsuka ◽

Masaki Takeuchi ◽

Tomoya Okabe ◽

Yoshiko Kamiya ◽

Ayami Hirata ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Fetal Liver ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Hematopoietic Organ ◽

Expression Levels ◽

Endothelial Progenitors

Abstract Blood cells and endothelia are believed to arise from their common progenitor hemangioblast. However, it still remains unknown how these lineages develop. Here we report the existence of two distinct precursors for hematopoietic stem cells (HSCs) and endothelial progenitors in murine fetal liver (FL). Podocalyxin-like protein 1 (PCLP1) is a member of the sialomucin family and was shown to be expressed in hemangioblasts in the aorta-gonad-mesonephros region in murine embryo. To further analyze the fates of hematopoietic/endothelial cells, we focused on embryonic day 14.5 (E14.5) FL, since it is a major hematopoietic organ during embryonic period. Based on the PCLP1 expression levels, E14.5 FL cells could be fractionated into four distinct populations. In vitro colony-forming assay and in vivo transplantation analysis revealed that lineage-committed progenitors with colony-forming activities and long-term repopulating hematopoietic stem cells (LTR-HSCs) were in PCLP1neg cells. PCLP1dull cells contained erythroid lineage-committed cells. Interestingly, while PCLP1med cells lacked colony-forming activities, they showed LTR-HSC activity in vivo. To further characterize these cell populations, we cultured them with OP9 stromal cells, since OP9 cells have been used to induce hematopoietic and endothelial lineages from embryonic stem cells. In co-culture with OP9 cells, PCLP1neg cells immediately generated blood cells with colony-forming activity but lacking in vivo hematopoietic activity, indicating that OP9 cells failed to support hematopoietic progenitor/HSCs. However, PCLP1med generated colony-forming hematopoietic progenitors with LTR-HSC activities in the presence of OP9 cells. These results indicated that PCLP1med cells contained stromal cell-dependent immature precursors for HSCs. PCLP1high cells did not express the hematopoietic markers or endothelial cell markers such as PECAM1 and VE-cadherin. However, they formed endothelia-like cell colonies which were highly proliferative and serially transferable in OP9 co-culture. Interestingly, the addition of vascular endothelial growth factor (VEGF) to the culture strongly induced the expression of PECAM1 and VE-cadherin in these colonies. PCLP1high cells contributed to PECAM1+ endothelium in several organs in vivo when transplanted to conditioned neonatal liver. Therefore, PCLP1high cells contained immature precursors for endothelial progenitors. These results indicate that PCLP1 expression levels distinguish previously unrecognized early precursors for HSCs and endothelial progenitors, which are distinct from hemangioblasts.

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Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow

Cytometry ◽

10.1002/(sici)1097-0320(19990901)37:1<60::aid-cyto7>3.0.co;2-u ◽

1999 ◽

Vol 37 (1) ◽

pp. 60-67

Author(s):

Christopher J. Donahue ◽

Christopher Fennie ◽

Ricardo Villacorta ◽

Hank La ◽

Laurence A. Lasky ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Hematopoietic Stem

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Different Roles of Glycosylphosphatidylinositol in Various Hematopoietic Cells as Revealed by a Mouse Model of Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood.v94.9.2963 ◽

1999 ◽

Vol 94 (9) ◽

pp. 2963-2970 ◽

Cited By ~ 69

Author(s):

Y. Murakami ◽

T. Kinoshita ◽

Y. Maeda ◽

T. Nakano ◽

H. Kosaka ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Cell Clone ◽

Early Stage ◽

Fetal Liver ◽

Cell Types ◽

Gpi Anchor ◽

Hematopoietic Stem ◽

Linked Gene

Abstract Patients with paroxysmal nocturnal hemoglobinuria (PNH) have one or a few clones of mutant hematopoietic stem cells defective in glycosylphosphatidylinositol (GPI) synthesis as a result of somatic mutation in the X-linked gene PIG-A. The mutant stem cell clone dominates hematopoiesis by a mechanism that is unclear. To test whether a lack of multiple GPI-anchored proteins results in dysregulation and expansion of stem cells, we generated mice in which GPI-anchor negative cells are present only in the hematopoietic system. We transplanted lethally irradiated mice with female fetal liver cells bearing one allele of the Piga gene disrupted by conditional gene targeting. Because of the X-chromosome inactivation, a significant fraction of the hematopoietic stem cells in fetal livers was GPI-anchor negative. In the transplanted mice, cells of all hematopoietic lineages contained GPI-anchor negative cells. The percentage of GPI-anchor negative cells was much higher in T lymphocytes including immature thymocytes than in other cell types, suggesting a regulatory role for GPI-anchored proteins at an early stage of T-lymphocyte development. However, the proportions of GPI-anchor negative cells in various blood cell lineages were stable over a period of 42 weeks, indicating thatPiga mutation alone does not account for the dominance of the mutant stem cells and that other phenotypic changes are involved in pathogenesis of PNH.

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Faculty Opinions recommendation of Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004105.47154 ◽

2002 ◽

Author(s):

Ana Cumano

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Hematopoietic Stem

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First blood: the endothelial origins of hematopoietic progenitors

Angiogenesis ◽

10.1007/s10456-021-09783-9 ◽

2021 ◽

Author(s):

Giovanni Canu ◽

Christiana Ruhrberg

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Cell Types ◽

Yolk Sac ◽

Vascular Endothelial ◽

Hematopoietic Stem ◽

Close Proximity ◽

Clinical Interest

AbstractHematopoiesis in vertebrate embryos occurs in temporally and spatially overlapping waves in close proximity to blood vascular endothelial cells. Initially, yolk sac hematopoiesis produces primitive erythrocytes, megakaryocytes, and macrophages. Thereafter, sequential waves of definitive hematopoiesis arise from yolk sac and intraembryonic hemogenic endothelia through an endothelial-to-hematopoietic transition (EHT). During EHT, the endothelial and hematopoietic transcriptional programs are tightly co-regulated to orchestrate a shift in cell identity. In the yolk sac, EHT generates erythro-myeloid progenitors, which upon migration to the liver differentiate into fetal blood cells, including erythrocytes and tissue-resident macrophages. In the dorsal aorta, EHT produces hematopoietic stem cells, which engraft the fetal liver and then the bone marrow to sustain adult hematopoiesis. Recent studies have defined the relationship between the developing vascular and hematopoietic systems in animal models, including molecular mechanisms that drive the hemato-endothelial transcription program for EHT. Moreover, human pluripotent stem cells have enabled modeling of fetal human hematopoiesis and have begun to generate cell types of clinical interest for regenerative medicine.

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