Simian Immunodeficiency Virus Infection of Hematopoietic Stem Cells and Bone Marrow Stromal Cells

Abstract The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.

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Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion

Transfusion ◽

10.1046/j.1537-2995.2002.00149.x ◽

2002 ◽

Vol 42 (7) ◽

pp. 921-927 ◽

Cited By ~ 39

Author(s):

Miki Yamaguchi ◽

Fumiya Hirayama ◽

Shinobu Wakamoto ◽

Mitsuhiro Fujihara ◽

Hideaki Murahashi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Stem

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Regulation of hematopoietic stem cells by bone marrow stromal cells

Trends in Immunology ◽

10.1016/j.it.2013.10.002 ◽

2014 ◽

Vol 35 (1) ◽

pp. 32-37 ◽

Cited By ~ 145

Author(s):

Bryan A. Anthony ◽

Daniel C. Link

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Stem

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Gene Transfer into Murine Hematopoietic Stem Cells and Bone Marrow Stromal Cells

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1990.tb24327.x ◽

1990 ◽

Vol 612 (1 Sixth Cooley') ◽

pp. 398-406 ◽

Cited By ~ 5

Author(s):

BARRY D. LUSKEY ◽

BING LIM ◽

JANE F. APPERLEY ◽

STUART H. ORKIN ◽

DAVID A. WILLIAMS

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Stem

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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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Angiopoietin-Like Protein 3 Supports the Activity of Mouse Hematopoietic Stem Cells in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v114.22.249.249 ◽

2009 ◽

Vol 114 (22) ◽

pp. 249-249

Author(s):

Junke Zheng ◽

HoangDinh Huynh ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Wild Type ◽

Hematopoietic Stem ◽

Hsc Niche

Abstract Abstract 249 The physiological role of Angiopoietin-like proteins (Angptls) in hematopoietic system is unknown. Here we showed that Angptl3 is expressed by both hematopoietic stem cells (HSCs) and bone marrow stromal cells. In particular, the expression of Angptl3 in the bone marrow stromal cells is significantly increased upon transplantation, suggesting that this protein may play an important role in the bone marrow under stress. We asked whether Angptl3 expression had a functional role in HSCs by utilizing mice ablated for Angptl3. Using Hoechst/pyronin Y staining and Brdu incorporation analysis, we found that HSCs in Angptl3-null mice exhibited significantly decreased quiescence compared to those in wild-type mice. To test the role of Angptl3 in the stress response of hematopoietic cells, we treated mice with 5-fluorouracil (5-FU), which is toxic to cycling cells and accelerates the entry of HSCs into the cell cycle. The survival of Angptl3-null mice was significantly lower than that of wild-type mice. To further identify the role of Angptl3 in stress response of HSCs, we examined whether Angptl3 affected DNA damage in HSCs upon transplantation. To this end, we transplanted WT bone marrow cells into lethally irradiated Angptl3 null recipients or WT mice. We found that HSCs in the Angptl3 null recipient mice had significantly increased gamma-H2AX foci compared to WT recipients, suggesting that Angptl3 protects HSCs from DNA damage. We further used the competitive reconstitution analysis to determine the roles of Angptl3 on HSC activity. Importantly, both Angptl3-null HSCs transplanted to wild-type recipients and wild-type HSCs transplanted to Angptl3-null recipients showed impaired repopulation. These results conclude that Angptl3 has both cell-autonomous and environmental effects that support the in vivo activity of HSCs. To identify the intracellular target of Angptl3 in HSCs, we performed DNA microarray and real-time RT-PCR analyses to compare the gene expression in HSCs isolated from WT and Angptl3 null mice. We found that Angptl3-null HSCs had increased expression of transcription factor Ikaros. Consistently, extrinsic treatment of HSCs by Angptl3 also suppressed the expression of Ikaros. Ikaros is a zinc finger transcription factor important for differentiation of lymphoid, myeloid, and erythroid cells, and its expression is low in multi-potent HSCs, but high in progenitors with lymphoid-myeloid potential. Since Angptl3 downregulates the expression of Ikaros in HSCs, we examined the effect of forced expression of Ikaros on HSC activities. Indeed, overexpression of Ikaros enhanced HSC cycling and DNA damage, and diminished their repopulation activity, indicating the downregulation of Ikaros by extrinsic Angptl3 maintains the stemness of HSCs. We studied the spatial relationship of Angptl3-expressing cells and the bone-marrow HSCs using immunohistochemical tools. We showed that 58.6% of Angptl3-producing cells were in contact with sinusoidal endothelial cells in bone marrow and that 60.8% of HSCs are adjacent to Angptl3-producing cells in the bone marrow. To directly test whether Angptl3-producing bone marrow stromal cells support HSC expansion, we co-cultured HSCs and CD45-SSEA4+ cells and used competitive reconstitution analysis to measure HSC activity. HSCs co-cultured with WT CD45-SSEA4+ cells had significantly increased repopulation relative to those co-cultured with Angptl3 null CD45-SSEA4+ cells (36% vs. 17%). This result demonstrated that bone marrow CD45-SSEA4+ cells support expansion of HSCs, and provided the functional evidence that Angptl3-producing stromal cells are a part of HSC niche in the bone marrow. Thus, Angptl3-producing cells are an important component of the HSC niche. Our experiments demonstrate a unique example of the extrinsic control of stemness by cell-autonomous effects from stem cells per se and by environmental effects from the niche cells. Disclosures: No relevant conflicts of interest to declare.

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An Alternative Growth Factor for Hematopoietic Stem Cells and Megakaryocytopoiesis.

Blood ◽

10.1182/blood.v104.11.2060.2060 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2060-2060 ◽

Cited By ~ 1

Author(s):

Mo Yang ◽

Karen Kwar Har Li ◽

C.K.Y. Chuen ◽

Ki Wai Chick ◽

Nga Hin Pong ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Growth Factor ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract The role of serotonin (5-hydroxytryptamine, 5-HT) on the regulation of blood stem cell proliferation and thrombopoiesis has not been recognized until 1996, when we reported that serotonin has a mitogenic effect on murine megakaryocytopoiesis via 5-HT2 receptors (Yang et al, Blood Coagul Fibrin 1996). Our study also indicated that the uptake ability of serotonin is well established in human megakaryoblasts (Yang et al, Int J Hematol, 1996). 5-HT 2A, 2B and 2C receptors were identified on human megakaryocytes and serotonin also promoted human megakaryocytopoiesis via these receptors (Yang et al, Blood, 2001; 2002 suppl). Thus, we established a new concept that serotonin is a growth factor for megakaryocytopoiesis (Yang et al, Blood, 2003 suppl). We further investigated the role of serotonin on human hematopoietic stem cells, bone marrow stromal cells and platelet formation. Serotonin (200 nM) significantly enhanced TPO, SCF plus FL -induced the ex vivo expansion of CD34+ cells, CD34+38- cells, CD41+61+ cells, CFU-GEMM and CFU-MK from cord blood CD34+ cells (MACS) (n=25) at day 8 (P<0.001). More significantly, serotonin enhanced the engraftment of human CD45+ cells, and their myeloid subsets CD33+ (p=0.05) in NOD/SCID mice. The expression of 5-HT 2A, 2B and 2C receptors was detectable in fresh CD34+ cells by RT PCR. These 5-HT receptors were further demonstrated in 1% of CD34+ cells by FACS. Our data also demonstrated that serotonin significantly stimulated the proliferation of bone marrow stromal cells in a dose-dependent manner (10–500 nM) in human CFU-F assay. A maximum stimulation was obtained at 200 nM of serotonin (n=5, p=0.03). The effect of serotonin was similar to that of PDGF and VEGF, but weaker than that of FGF-2 at their optimal dose. Serotonin also significantly enhanced FGF-2, PDGF or VEGF -induced CFU-F formation (p<0.05). 5-HT 2A, 2B and 2C receptors were also demonstrated in bone marrow stromal cells by RT-PCR and around 1% positive confirmed by FACS. Results on thrombopoiesis showed that 5-HT2A, 2B and 2C receptors were expressed strongly (80–99%) on MB megakaryocytes and MK cell lines. Serotonin also increased the size of cultured megakaryocyte suggesting it has a promoting effect on megakaryocyte maturation. Ketanserin, a 5-HT2B receptor antagonist, was showed in the same study to block the mitogenic effect on megakaryocyte differentiation. Serotonin also has an effect on actin re-organization in Meg-01 cells. We have provided the evidence of serotonin as a growth factor for blood stem cells and MK cells. Based on this concept, alternative drug could be developed for the treatment of thrombocytopenia.

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