Endothelial Cells Are Not Derived from Hematopoietic Precursor Cells.

Abstract Endothelial outgrowth cells (EOC) can be generated from mononuclear blood cells. Based on proliferative and functional characteristics, EOC were claimed to derive from an immature endothelial progenitor cell or angioblast. Several investigators have claimed that these cells constitute a subpopulation of CD34+ hematopoietic stem cells(HSC). However, the EOC-precursor is not well defined and its nature remains elusive. Methods and results: Umbilical cord blood CD34+ cells were sorted into a small (< 1 %) CD34+CD45− non-hematopoietic cell fraction (purity > 99.5%) and CD34+CD45+ HSC (purity > 99.2 %) (n=5). The cell fractions were cultured separately in EBM2/EGM2 medium (Cambrex, Verviers, Belgium) onto gelatine coated 24 wells. EOC were exclusively derived from the CD34+CD45− cell fraction and not from the CD45+ HSC. We further analysed the CD34+CD45− cell fraction for expression of endothelial progenitor genes. Analysis showed the presence of VEGFR2, VE-Cadherine and CD146 on the CD34+CD45− precursor population whereas CD45+ HSC were consistantly negative for these markers. CD133, which was claimed to be a marker for endothelial progenitors was negative on the CD34+CD45− cells. No VEGFR2+ CD133+ cells could be detected either by flowcytometry or at the mRNA level. In adult bone marrow, EOC only derived from CD45− CD31+ cells, and not from the CD45+ HSC or CD45− CD31− mesenchymal cells. CD34+CD45+ HSC or CD14+ CD45+ monocytes generated under the same conditions large flat adherent cells positive for CD31, LDL uptake and the lectin UEA-1. On RT-PCR and real time RT-PCR analysis, cells were positive for VEGFRII, CD146 and VE cadherin. However, membrane staining was consistently negative for VE-cadherin on flowcytometric analysis and positive for monocytic markers such as CD14 and CD45. In functional assays, the majority of the cells were shown to be phagocytic and were unable to form vascular tubes in the matrigel angiogenesis assay. These data demonstrate that monocytes may acquire a phenotype in vitro which is difficult to discriminate from endothelial cells. Conclusion : Endothelial cell generated in vitro from cord blood or bone marrow derive from a CD45− nonhematopoietic precursor.

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Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽

10.1182/blood.v108.11.1652.1652 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1652-1652

Author(s):

Patrick Ziegler ◽

Steffen Boettcher ◽

Hildegard Keppeler ◽

Bettina Kirchner ◽

Markus G. Manz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Human Mesenchymal Stem Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

Blood ◽

10.1182/blood.v110.11.4101.4101 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4101-4101

Author(s):

Wen-ming Chen ◽

Zi-xing Chen ◽

Jian-nong Cen ◽

Jun He ◽

Xiao-li Jiao ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Biological Characteristics ◽

Bone Marrow Mononuclear Cell ◽

Feeder Layer ◽

Rt Pcr ◽

Osteoblastic Cell Line ◽

Hematopoietic Stem ◽

Gm Csf

Abstract It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

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Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽

10.1182/blood.v116.21.2433.2433 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2433-2433

Author(s):

Medhat Shehata ◽

Rainer Hubmann ◽

Martin Hilgarth ◽

Susanne Schnabl ◽

Dita Demirtas ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

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Real-Time RT-PCR Analysis of Individual Osteolineage Cells within the Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v118.21.2389.2389 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2389-2389

Author(s):

Lev Silberstein ◽

Masatake Osawa ◽

Charles Lin ◽

Peter Kharchenko ◽

Cristina Lo Celso ◽

...

Keyword(s):

Bone Marrow ◽

Pcr Analysis ◽

Rna Seq ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Chi Squared ◽

The Individual ◽

Osteolineage Cells ◽

Angiopoietin 1

Abstract Abstract 2389 Osteolineage cells (OLCs) have been shown to participate in a regulatory bone marrow microenvironment for the hematopoietic stem and progenitor cells (HSPCs) – the endosteal niche. Our previous experiments using live animal imaging have demonstrated that single transplanted HSPCs preferentially home in close proximity to the individual OLCs. We hypothesized that these HSPC-proximal cells represent a distinct subpopulation of OLCs, which is specifically involved in a non-cell autonomous regulation of HSPC quiescence and self-renewal. To test this hypothesis, we developed a novel experimental platform, which allows visualization of HSPC-OLC cell pairs in-vivo and retrieval of the individual OLCs for molecular analysis. We intravenously injected DiI labeled adult bone marrow-derived FACS-sorted Lin−Sca1+c-kit+CD34−Flk2− HSPCs into irradiated newborn collagen 2.3GFP mouse recipients; in this transgenic strain, the majority of the OLCs are labeled with green fluorescent protein (GFP). 48 hours later, we sacrificed the animals and obtained fresh unfixed sections of femoral trabecular bone. Using a combination of differential interference contrast fluorescent microscopy, in-situ enzymatic digestion and micromanipulation, we harvested individual GFP-positive OLCs located within 2 cell diameters (“niche” OLCs) or greater than 5 cell diameters (“control” OLCs) from single DiI-bright HSPCs. Following reverse transcription and cDNA amplification with 29 cycles of PCR, as per the single cell RNA-Seq protocol (Tang et al, Nature Protocols 2010), we performed real-time RT-PCR analysis of 31 samples – 15 niche cells and 16 controls - for the OLC signature genes (osteocalcin, osterix) and for the genes implicated in playing a functional role in the HSPC-OLC cell interaction (osteopontin, CXCL12, angiopoietin 1). Transcripts for GAPDH, collagen 1 and GFP served as positive controls for the amplification. As expected, all cells were positive for GFP and over 85% cells expressed collagen 1. Osteopontin and CXCL12 were expressed at a similar level and frequency in the niche and control OLCs. However, we found that angiopoietin 1 transcripts were detected exclusively in the niche OLCs (3/15 versus 0/16, p <0.05 by Chi-squared). Moreover, niche OLCs were enriched for the osterix-positive cells (7/15 versus 2/16, p <0.05 by Chi-squared) and expressed a lower level of osteocalcin, as normalized for GAPDH expression (1.13 vs. 0.97, p< 0.05 by t-test). Our results suggest that niche OLCs may have a distinct molecular signature and reside within a population of very immature OLCs, as evidenced by the osterix + osteocalcin low phenotype. Further unbiased transcriptome characterization of these cells using genome-wide RNA-Seq assay is therefore likely to provide more evidence in support of our hypothesis and reveal novel non-cell autonomous regulators of HSPC quiescence. To our knowledge, this approach represents the first attempt to define molecular heterogeneity in-vivo at a single cell level using the micro-anatomical relationship between two heterologous cell types. Disclosures: Scadden: Fate Therapeutics: Equity Ownership.

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Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽

10.1182/blood-2019-122449 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2488-2488 ◽

Cited By ~ 1

Author(s):

José Gabriel Barcia Durán

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Fluorescent Microscopy ◽

Hematopoietic Stem ◽

Interleukin Receptors ◽

Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

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RUNX1 Expression Characterizes the Endothelial Cells from the Spleen and Bone Marrow of Patients with Primary Myelofibrosis

Blood ◽

10.1182/blood-2018-99-114564 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5486-5486

Author(s):

Rita Campanelli ◽

Margherita Massa ◽

Laura Villani ◽

Paolo Catarsi ◽

Carlotta Abbà ◽

...

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Cord Blood ◽

Network Formation ◽

Splenic Vein ◽

Primary Myelofibrosis ◽

Laser Scanning Microscopy ◽

Hematopoietic Stem ◽

Spleen Tissue ◽

Runx1 Expression

Abstract Background. Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder characterized by extramedullary haematopoiesis and abnormal neoangiogenesis in both the bone marrow (BM) and the spleen. We previously provided evidence that endothelial cells (ECs) from either the spleen or the splenic vein of PMF patients frequently share the JAK2V617F mutation with the hematopoietic malignant cells. More recently, we confirmed this observation also in BM-derived ECs of PMF patients. The mechanism underlying this phenomenon remains, however, not yet clarified. RUNX1 is a critical regulator of hematopoiesis, required for hematopoietic stem cell (HSC) generation and function. In human embryo, it is expressed in all emerging HSCs and progenitors and it is a necessary transcription factor for endothelial to hematopoietic transition. In the adult humans it is expressed in all blood cells, in decreasing intensity according to the maturation status, except erythrocytes. In angiogenesis, it induces endothelial differentiation and maturation as well as vascular network formation by promoting expression of VE-cadherin. Finally, it is involved in retinal aberrant neoangiogenesis. Aim. To assess if neoangiogenetic activity observed in spleen and BM of PMF patients is associated with RUNX1 expression in ECs. Patients and Methods. Paraffin-embedded BM sections from patients with MF (n=3), and from patients with lymphomas (named as CTRLs), who underwent BM biopsy for disease staging (n=2), were used for immunostaining analysis. Spleens samples were collected from 3 patients with PMF, who received splenectomy for clinical reasons, and from 2 healthy subjects (HS), who underwent surgery after traumatic damage. Fresh spleen samples were embedded in OCT, and then snap-frozen and stored in liquid nitrogen. Endothelial colony forming cells (ECFCs) were obtained from peripheral blood (n=1 PMF, n=1 HS), PMF spleen tissue (n=1) and cord blood (n=1), according to Ingram et al (Blood 2004;104:2752) and cytospun on slides at confluence. ECFCs, BM and spleen sections were stained with antibodies directed against RUNX1 and VE-cadherin. The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGEJ software. We evaluated 10 fields for each BM and spleen section and measured the number of RUNX1-positive vessels/total vessels. We considered positive a vessel that contained at least one RUNX1-positive cell. The results are shown as mean ± SD. Results. Immunofluorescence staining of spleen and BM sections confirmed the presence of increased neoangiogenetic processes in samples from PMF patients with respect to CTRLs. The staining of BM and spleen sections obtained from PMF patients with antibodies anti-RUNX1 and anti-VE-cadherin (endothelial marker) allowed the detection of RUNX1+VE-cadherin- cells in the parenchima of BM and spleen (Figure 1A, 1C), occasionally in perivascular position (Figure 1A, arrowhead). Interestingly, in all patients analyzed, we could detect RUNX1+VE-cadherin+ cells in both the parenchima and in the vessels both in the BM and the spleen (Figure 1A, 1C, arrow). In BM, 22 ± 12% of the vessels had at least one double positive cell in the microvessel wall; in spleen tissue the percentage of RUNX1+ ECs increased to 60 ± 13%. We detected RUNX1+VE-cadherin- cells only in the BM parenchima of CTRLs but not in the spleen of HS, whereas RUNX1+VE-cadherin+ cells were never observed in the vessels of neither the spleen nor the BM (Figure 1B, 1D) of HS and CTRLs, respectively. The circulating ECFCs obtained from both PMF and HS were, as expected, VE-cadherin+ but did not express RUNX1; however, 30% of spleen-derived-ECFCs of the PMF patient were RUNX1+. In cord blood-derived ECFCs a small percentage (3%) of RUNX1+ cells was observed. Conclusions. Our data show a selective expression of RUNX1 in splenic- and BM-ECs of PMF patients, suggesting that activation of RUNX1 expression could be associated with the neoangiogenetic processes that characterize the disease. The expression of RUNX1 in ECFCs from spleen but not in their circulating counterpart suggests a role for the splenic microenvironment in determining RUNX1 expression in ECs. Further studies are ongoing to assess the genotype of RUNX1+ ECs of PMF patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Influence of the endothelial cells on the erythroid differentiation of umbilical cord blood hematopoietic stem cells in vitro

Proceedings of the National Academy of Sciences of Belarus Medical series ◽

10.29235/1814-6023-2020-17-1-78-86 ◽

2020 ◽

Vol 17 (1) ◽

pp. 78-86

Author(s):

A. S. Voytehovich ◽

E. V. Vasina ◽

V. S. Kastsiunina ◽

I. N. Seviaryn ◽

N. V. Petyovka

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Vascular Niche

The objective is to study the effect of umbilical cord blood endothelial cells on the hematopoietic cells growth and the maturation in the erythroid direction in co-culture, as well as the expression of adult and fetal hemoglobin genes during erythroid differentiation under the conditions of vascular niche modeling in vitro. We used the following research methods: cultural, flow cytometry, real-time PCR and morphological analysis. We have developed the method of hematopoietic cord blood stem cells erythroid differentiation in co-culture using cord blood endothelial cell progenitors. CD34+CD31+CD144+CD105+CD90–CD45– progenitors of endothelial cells stimulate the erythroid differentiation of hematopoietic CD34+ cord blood cells and the growth of erythroid progenitors in co-culture from the 4th to 11th day in the presence of the stem cell factor, the erythropoietin and the fibroblast growth factor-2. The in vitro modeling of the vascular niche increases the mature CD36–CD235a+ erythroid cells 2.5 times higher than those in the liquid culture. The microenvironment of endothelial cells does not affect the level and expression ratio of fetal and adult hemoglobin during the erythroid differentiation in vitro.

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Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Blood ◽

10.1182/blood.v88.7.2510.bloodjournal8872510 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2510-2516 ◽

Cited By ~ 11

Author(s):

M Holberg-Petersen ◽

H Rollag ◽

S Beck ◽

I Overli ◽

G Tjonnfjord ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Clinical Isolate ◽

Human Cytomegalovirus ◽

Mononuclear Cells ◽

Colony Growth ◽

Structural Protein ◽

Hematopoietic Stem ◽

Cd34 Cells

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

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