scholarly journals Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2844-2850 ◽  
Author(s):  
N Yanai ◽  
C Sekine ◽  
H Yagita ◽  
M Obinata
Keyword(s):  
Progenitor Cells ◽  
Adhesion Molecules ◽  
Stromal Cells ◽  
Ligand Molecule ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Activation Antigen

Abstract Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver

Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 379-384
Author(s):  
O. Ohneda ◽  
N. Yanai ◽  
M. Obinata
Keyword(s):  
Progenitor Cells ◽  
Stromal Cells ◽  
Close Contact ◽  
Fetal Liver ◽  
Rapid Expansion ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Semisolid Medium ◽  
Mouse Fetus ◽  
Erythroid Progenitor Cells

Mouse stromal cell lines (FLS lines), established from the livers of 13-day gestation mouse fetus, supported the proliferation and differentiation of the erythroid progenitor cells from mouse fetal livers and bone marrow in a semisolid medium in the presence of erythropoietin. A large erythroid colony of over 1000 benzidine-positive erythroid cells was developed from a single erythroid progenitor cell on the FLS cell layer after 4 days of culture. When in close contact with the layer, the erythroid progenitor cells divided rapidly with an average generation time of 9.6 h and mature erythroid cells, including enucleated erythrocytes, were produced. The present studies demonstrate that the microenvironment created by the stromal cells can support the rapid expansion of erythropoietic cell population in the fetal liver of mice.

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.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3211-3211
Author(s):  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Kazuyuki Murase ◽  
Satoshi Iyama ◽  
Tsutomu Sato ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Production System ◽  
Ex Vivo ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Human Stromal Cells

Abstract Abstract 3211 We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells. Disclosures: No relevant conflicts of interest to declare.

Analysis of Mesenchymal Stromal Cells (MSC) and Their Interactions with CD34 Stem and Progenitor Cells in Patients with Myelodysplastic Syndromes (MDS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2393-2393
Author(s):  
Stefanie Geyh ◽  
Ron Patrick Cadeddu ◽  
Julia Fröbel ◽  
Ingmar Bruns ◽  
Fabian Zohren ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myelodysplastic Syndromes ◽  
Stromal Cells ◽  
Median Number ◽  
Lower Number ◽  
Differentiation Potential ◽  
Receptor Ligand ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 2393 Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders and research in this field has mainly focused on hematopoietic stem and progenitor cells (HSPC). Still, recent data from mouse models indicate that the bone marrow (BM) microenvironment might be involved in the pathogenesis MDS (Raaijmakers et al., 2010). The role of mesenchymal stromal cells (MSC) in particular as a key component of the BM microenvironment remains elusive in human MDS and data so far are controversial. Design/Methods: We therefore investigated MSC and immunomagnetically enriched CD34+ HSPC from BM of 42 untreated patients (pts) with MDS (12 RCMD, 12 RAEB, 12 sAML, 3 del5q, 1 CMML-1, 1 MDS hypocellular, 1 MDS unclassifiable according to WHO) and age-matched healthy controls (HC, n=13). MSC were examined with regard to growth kinetics, morphology and differential potential after isolation and expansion according standard procedures in line with the international consensus criteria (Dominici et al., 2006). Furthermore corresponding receptor-ligand pairs on MSC and CD34+ cells (Kitlg/c-kit; CXCL12/CXCR4; Jagged1/Notch1; Angpt1-1/Tie-2; ICAM1/LFA-1) were investigated by RT-PCR. Results: In MDS, the colony forming activity (CFU-F) of MSC was significantly reduced in comparison to HC (median number of colonies per 1×107MNC in MDS: 8, range 2–74 vs. HC: 175, 10–646, p=0.003) and this was also true when looking at the different subtypes (RCMD median: 16, p=0.04; RAEB median: 8, p=0.31; sAML median: 26, p=0.02). According to this, MSC from pts with RCMD and del5q could only be maintained in culture for a lower number of passages (median number of passages: MDS 3 passages, range 1–15; HD 14 passages, range 8–15, p=0.01), had a lower number of cumulative population doublings (CPD) and needed a longer timer to reach equivalent CPD (MDS median: 18,16 CPD, HD median: 33,68 CPD, p=0,0059). All types of MDS-MSC showed an abnormal morphology, while an impaired osteogenic differentiation potential was exclusively observed in pts with RCMD. These findings of an altered morphology together with a diminished growth and differentiation potential prompted us to test, whether the interaction between MSC and CD34+ HSPC in BM of pts with MDS was also disturbed. On the MDS-MSC, we found a significant lower expression of Angpt1 in pts with RAEB (3.5-fold, p=0.01) and del5q (4.9-fold, p=0.009) compared to HD. The expression of CXCL12 (2.5-fold, p=0.057) and jagged1 was reduced in trend in MSC from pts with MDS, while no differences were observed with regard to the expression of kitlg and ICAM1. When looking on CD34+ cells, we found a significantly reduced expression of CXCR4 (RCMD 2.5-fold, p=0.02; RAEB 2.46-fold, p=0.02), notch1 (RCMD 6-fold, p=0.04) and Tie-2 (RAEB 5.91-fold, p=0.02) in pts with MDS, while LFA-1 was overexpressed in pts with RAEB (2.6-fold, p=0.036). Conclusion: Taken together, our data indicate that MSC from pts with MDS are structurally altered and that the crosstalk between CD34+ HSPC and MSC in the BM microenvironment of pts with MDS might be deregulated as a result of an abnormal expression of relevant receptor-ligand pairs. Ongoing research is required to corroborate these findings and to definitely address their functional relevance for the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Neuromedin U Peptide Activates STAT5 and S6 in a JAK-2 Dependent Manner and Promotes Erythroid Cell Growth in Primary Erythroid Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1241-1241
Author(s):  
Rebecca Lenzo ◽  
Martha Dua-Awereh ◽  
Martin Carroll ◽  
Susan E. Shetzline
Keyword(s):  
Cell Growth ◽  
Progenitor Cells ◽  
Surrogate Marker ◽  
Janus Kinase ◽  
Erythroid Cell ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Pi3k Activation

Abstract Abstract 1241 Erythropoiesis is a multi-step process during which hematopoietic stem cells terminally differentiate into red blood cells (RBCs). Erythropoietin (EPO) is the only known cytokine regulator of terminal erythroid differentiation. Previously, we reported that the neuropeptide, neuromedin U (NmU), which interacts with NmU receptor type 1 (NMUR1), functions as a novel extracellular cofactor with EPO to promote the expansion of early erythroblasts, which are CD34−, CD71+, glycophorin A (GlyA)dim(Gambone et al, Blood. 2011). Here, we describe studies to understand the mechanism whereby NmU augments EPO effects on erythroid cell growth. EPO triggers Janus kinase (Jak)-2 dependent activation of signal transducer and activator of transcription (STAT) 5 and phosphatidylinositol 3-kinase (PI3K) to promote the proliferation and/or survival of erythroid progenitor cells. We hypothesized that NmU peptide would cooperate with EPO to promote the proliferation of early erythroblasts through STAT5 and/or PI3K activation. To address this hypothesis, we cultured primary human CD34+ cells in 2-stage liquid culture with IL-3, IL-6, and stem cell factor (SCF) from day 0 to day 6. On day 6, 2U/mL of EPO was added, and the cells were cultured for an additional 5 days to expand erythroid progenitors. On day 11, cells were briefly serum starved and then stimulated with EPO and/or NmU in the absence or presence of a Jak-1/2 inhibitor. Activation of STAT5 and S6, a surrogate marker for PI3K activation, were assessed by phospho-flow in ERY3 (CD34−, CD71+, GlyA+) and ERY4 (CD34−, CD71dim, GlyA+) cells. As expected, EPO alone activated STAT5 and S6 in ERY3 cells only, and the presence of a Jak-1/2 inhibitor diminished STAT5 activation. Interestingly, STAT5 and S6 were activated by NmU peptide alone in ERY3 and ERY4. Surprisingly, in the presence of a Jak-1/2 inhibitor, NmU peptide, which binds to NMUR1 a G-protein coupled receptor, did not activate STAT5 or S6 in ERY3 or 4 cells, suggesting that NmU functions through a JAK kinase in erythroid cells. No additive or synergistic activation of STAT5 and S6 is observed in the presence of both EPO and NmU peptide when EPO was used at a dose of 2 U/mL. The mechanism whereby NmU activates a JAK dependent signaling pathway is under investigation. Preliminary evidence suggests that EPO induces the physical association of NMUR1 with EPO receptor (EPOR). Taken together, we propose that NmU is a neuropeptide expressed in bone marrow cells that cooperates to regulate erythroid expansion during early erythropoiesis through the activation of cytokine receptor like signaling pathways and perhaps through direct interaction with EPOR. NmU may be useful in the clinical management of anemia in patients unresponsive to EPO or other erythroid-stimulating agents. Disclosures: No relevant conflicts of interest to declare.

Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary Cultured Human Erythroid Precursor Cells From Apoptosis

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1568-1577 ◽  
Author(s):  
Yoshihito Haseyama ◽  
Ken-ichi Sawada ◽  
Atsushi Oda ◽  
Kazuki Koizumi ◽  
Hina Takano ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Maximal Effect ◽  
Erythroid Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Erythroid Progenitor Cells ◽  
Dependent Cell ◽  
Phosphatidylinositol 3

Little is known about the physiologic role of phosphatidylinositol 3-kinase (PI-3K) in the development of erythrocytes. Previous studies have shown that the effects of the PI-3K inhibitor wortmannin on erythropoietin (EPO)-dependent cell lines differed depending on the cell type used. Wortmannin inhibited EPO-induced differentiation of some cell lines without affecting their proliferation; however, the EPO-induced proliferation of other cell lines was inhibited by wortmannin. In neither case were signs of apoptosis observed. We have previously reported that signaling in highly purified human colony forming units-erythroid (CFU-E), generated in vitro from CD34+ cells, differed from that in EPO-dependent cell lines. In the current study, we examined the effects of a more specific PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002 dose-dependently inhibits the proliferation of erythroid progenitor cells with a half-maximal effect at 10 μmol/L LY294002. LY294002 at similar concentrations also induces apoptosis of these cells, as evidenced by the appearance of annexin V–binding cells and DNA fragmentation. The steady-state phosphorylation of AKT at Ser-473 that occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are specific. Interestingly, the acceleration of apoptosis by LY294002 was observed in the presence or absence of EPO. Further, deprivation of EPO resulted in accelerated apoptosis irrespective of the presence of LY294002. Our study confirms and extends the finding that signaling in human primary cultured erythroid cells is significantly different from that in EPO-dependent cell lines. These data suggest that PI-3K has an antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive and one that is EPO-dependent and at least partly insensitive to LY294002.

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