Effects of mouse fetal liver cell culture density on hematopoietic cell expansion in three-dimensional cocultures with stromal cells

2021 ◽  
pp. 039139882199637
Author(s):  
Hirotoshi Miyoshi ◽  
Kenji Abo ◽  
Daiki Hosoya ◽  
Kazuyuki Matsuo ◽  
Yoshio Utsumi
Keyword(s):  
Cell Lines ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Three Dimensional ◽  
Porous Polymer ◽  
Hematopoietic Stem ◽  
Culture Density ◽  
Effective Expansion ◽  
Cell Layers

Objective: An effective ex vivo expansion system of primitive hematopoietic cells (HCs) is required for wider application of hematopoietic stem cell transplantation. In this study, we examined effects of culture density on mouse fetal liver cells (FLCs) used as an HC source for the expansion of primitive HCs in three-dimensional (3D) cocultures with two kinds of mouse stromal cell lines (OP9 or C3H10T1/2). Materials and methods: FLCs were seeded at different densities (1, 2, and 10 × 107 cells/cm3) into porous polymer scaffolds with or without stromal cell layers and HCs were expanded in the cultures for 2 weeks without exogenous cytokines. Results: Differential effects of culture density on HC expansion were observed between cocultures and solitary FLC controls. In stromal cell cocultures, high expansion of HCs was achieved when FLCs were seeded at low densities. In contrast, the expansion in the controls was enhanced with increasing culture densities. With respect to expansion of primitive HCs existing in the FLCs, cocultures with C3H10T1/2 cells were superior to those with OP9 cells with a 29.3-fold expansion for c-kit+ hematopoietic progenitor cells and 8.3-fold expansion for CD34+ hematopoietic stem cells. In the controls, HC expansion was lower than in any cocultures, demonstrating the advantages of coculturing for HC expansion. Conclusion: Stromal cell lines are useful in expanding primitive HCs derived from FLCs in 3D cocultures. Culture density is a pivotal factor for the effective expansion of primitive HCs and this effect differs by culture condition.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals In Vitro Maintenance of Highly Purified, Transplantable Hematopoietic Stem Cells

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4337-4347 ◽  
Author(s):  
Kateri A. Moore ◽  
Hideo Ema ◽  
Ihor R. Lemischka
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity

Abstract The cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity. In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells. This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.

scholarly journals Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1167-1175 ◽  
Author(s):  
Ben S. Lam ◽  
Cynthia Cunningham ◽  
Gregor B. Adams
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Cell Activity ◽  
Calcium Sensing Receptor ◽  
Hematopoietic Stem ◽  
Calcium Sensing ◽  
Endosteal Niche

Abstract The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist, Cinacalcet, we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro, including growth in stromal cell cocultures, adhesion to extracellular matrix molecules such as collagen I and fibronectin, and migration toward the chemotactic stimulus, stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing, CXCR4-mediated lodgment at the endosteal niche, and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM.

scholarly journals Ex Vivo-Generated CD36+ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

2007 ◽  
Vol 82 (5) ◽  
pp. 2470-2476 ◽  
Author(s):  
Susan Wong ◽  
Ning Zhi ◽  
Claudia Filippone ◽  
Keyvan Keyvanfar ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Real Time ◽  
Cell Lines ◽  
Ex Vivo ◽  
Parvovirus B19 ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Human Virus ◽  
Erythroid Progenitor Cells ◽  
Large Numbers

ABSTRACT The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Expansion of mouse hematopoietic stem/progenitor cells in three-dimensional cocultures on growth-suppressed stromal cell layer

2019 ◽  
Vol 42 (7) ◽  
pp. 374-379 ◽  
Author(s):  
Hirotoshi Miyoshi ◽  
Chiaki Sato ◽  
Yuichiro Shimizu ◽  
Misa Morita
Keyword(s):  
Progenitor Cells ◽  
Mitomycin C ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Three Dimensional ◽  
Hematopoietic Cells ◽  
Liver Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors

1993 ◽  
Vol 54 (6) ◽  
pp. 1017-1021 ◽  
Author(s):  
Sigrun Gabius ◽  
Ralf Wawotzny ◽  
Sabine Wilholm ◽  
Ulrikc Martin ◽  
Bernhard Wörmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Stromal Cell ◽  
Bone Marrow Stromal Cell ◽  
Marrow Stromal Cell ◽  
Human Lymphoid Cell ◽  
Cell Layers ◽  
Sugar Receptors ◽  
Lymphoid Cell Lines

scholarly journals Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver

2020 ◽  
Author(s):  
Amina Mohammadalipour ◽  
Miguel F. Diaz ◽  
Sumedha Pareek ◽  
Pamela L. Wenzel
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Stem Cell Homing

Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3568-3568
Author(s):  
Mattias Magnusson ◽  
Melissa Romero ◽  
Sacha Prashad ◽  
Ben Van Handel ◽  
Suvi Aivio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Placenta ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Cell Types ◽  
First Trimester ◽  
Hematopoietic Stem ◽  
Human Hscs

Abstract Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.

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