The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

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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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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In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽

10.1182/blood.v78.12.3155.3155 ◽

1991 ◽

Vol 78 (12) ◽

pp. 3155-3161 ◽

Cited By ~ 1

Author(s):

RM Schwartz ◽

SG Emerson ◽

MF Clarke ◽

BO Palsson

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Culture Medium ◽

Culture Conditions ◽

Hematopoietic Growth Factors ◽

Hematopoietic Stem ◽

Gm Csf ◽

Medium Exchange ◽

Proliferation And Differentiation

Abstract We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

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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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Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽

10.1182/blood.v122.21.143.143 ◽

2013 ◽

Vol 122 (21) ◽

pp. 143-143 ◽

Cited By ~ 4

Author(s):

Saar Gill ◽

Sarah K Tasian ◽

Marco Ruella ◽

Olga Shestova ◽

Yong Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Research Funding ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Gm Csf ◽

Nsg Mice ◽

Cd34 Cells ◽

Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

Blood ◽

10.1182/blood.v112.11.2440.2440 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2440-2440

Author(s):

Nils Heinrich Thoennissen ◽

Tadayuki Akagi ◽

Sam Abbassi ◽

Daniel Nowak ◽

Ann George ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Transcription Factors ◽

Bone Marrow Cells ◽

Double Knockout ◽

Hematopoietic Stem ◽

Gm Csf ◽

Marrow Cells

Abstract CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

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Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

Journal of Immunology Research ◽

10.1155/2016/5479013 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Yu Han ◽

Huaquan Wang ◽

Zonghong Shao

Keyword(s):

Myelodysplastic Syndrome ◽

Ex Vivo ◽

Regulatory Protein ◽

Control Group ◽

Phagocytic Capacity ◽

Hematopoietic Stem ◽

Gm Csf ◽

Increased Risk ◽

Macrophage Colony

Background. The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. We analyzed the role of macrophages from MDS patients in vitro. Methods. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Phagocytic capacity of macrophages was measured with carboxyfluorescein succinimidyl ester and fluorescent microspheres. CD206 and signal regulatory protein alpha (SIRPα) on macrophages were detected by flow cytometry. Inducible nitric oxide synthase (iNOS) was measured by ELISA method. Results. Compared with normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and SIRPα were lower. Levels of iNOS secreted by macrophages in MDS were increased. Conclusions. Monocyte-derived macrophages are impaired in myelodysplastic syndromes.

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In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia

Blood ◽

10.1182/blood.v78.9.2203.2203 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2203-2210 ◽

Cited By ~ 55

Author(s):

GP Bagnara ◽

G Zauli ◽

L Vitale ◽

P Rosito ◽

V Vecchi ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Gm Csf ◽

Diamond Blackfan Anemia ◽

Clear Explanation ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Abstract Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte- conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.

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Endothelial Cells Are Not Derived from Hematopoietic Precursor Cells.

Blood ◽

10.1182/blood.v108.11.1815.1815 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1815-1815

Author(s):

Frank Timmermans ◽

Magda De Smedt ◽

Robrecht Raedt ◽

Jean Plum ◽

Bart Vandekerckhove

Keyword(s):

Bone Marrow ◽

Endothelial Cells ◽

Cord Blood ◽

Mrna Level ◽

Cell Fraction ◽

Pcr Analysis ◽

Rt Pcr ◽

Endothelial Progenitor ◽

Hematopoietic Stem

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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In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia

Blood ◽

10.1182/blood.v78.9.2203.bloodjournal7892203 ◽

1991 ◽

Vol 78 (9) ◽

pp. 2203-2210

Author(s):

GP Bagnara ◽

G Zauli ◽

L Vitale ◽

P Rosito ◽

V Vecchi ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Gm Csf ◽

Diamond Blackfan Anemia ◽

Clear Explanation ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Diamond-Blackfan anemia (DBA) is a congenital red blood cell aplasia. No clear explanation has been given of its defective erythropoiesis, although different humoral or cellular inhibitory factors have been proposed. To clarify the nature of this defect we studied the effect of several human recombinant growth factors on an enriched CD34+ population obtained from the bone marrow of 10 DBA patients. We observed a defect underlying the early erythroid progenitors, which were unresponsive to several growth factors (erythropoietin, interleukin-3 [IL-3], IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF], erythroid potentiating activity), either alone or in association. The production of cytokines was not impaired, and high levels of IL-3 and GM-CSF were found in phytohemagglutinin-leukocyte- conditioned medium (PHA-LCM) when tested with a sensitive biologic assay on the M-07E cell line. Hematopoietic stem cells in DBA patients may be induced to differentiate to the granulocyte megakaryocyte, but not the erythroid compartment, as shown after CD34+ cell preincubation with IL-3. Addition of the stem cell factor to IL-3 and erythropoietin induces a dramatic in vitro increase in both the number and the size of BFU-E, which also display a normal morphologic terminal differentiation.

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In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽

10.1182/blood.v78.12.3155.bloodjournal78123155 ◽

1991 ◽

Vol 78 (12) ◽

pp. 3155-3161 ◽

Cited By ~ 50

Author(s):

RM Schwartz ◽

SG Emerson ◽

MF Clarke ◽

BO Palsson

Keyword(s):

Bone Marrow ◽

Growth Factors ◽

Culture Medium ◽

Culture Conditions ◽

Hematopoietic Growth Factors ◽

Hematopoietic Stem ◽

Gm Csf ◽

Medium Exchange ◽

Proliferation And Differentiation

We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

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