Selection of Common Markers for Bone Marrow Stromal Cells from Various Bones Using Real-Time RT-PCR: Effects of Passage Number and Donor Age

2007 ◽  
Vol 13 (10) ◽  
pp. 2405-2417 ◽  
Author(s):  
Akira Igarashi ◽  
Kazumi Segoshi ◽  
Yuhiro Sakai ◽  
Haiou Pan ◽  
Masami Kanawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Donor Age ◽  
Rt Pcr ◽  
Passage Number ◽  
Selection Of

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

2002 ◽  
Vol 85 (4) ◽  
pp. 737-746 ◽  
Author(s):  
Oliver Frank ◽  
Manuel Heim ◽  
Marcel Jakob ◽  
Andrea Barbero ◽  
Dirk Schäfer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Pcr Analysis ◽  
Rt Pcr

Isolation Yield and Osteogenic Potential of Human Bone Marrow Stromal Cells are Maintained Irrespective of Age or Gender

2007 ◽  
Vol 361-363 ◽  
pp. 1149-1152
Author(s):  
Jeong Joon Yoo ◽  
Jeon Hyun Bang ◽  
Kyung Hoi Koo ◽  
Kang Sup Yoon ◽  
Hee Joong Kim
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Mononuclear Cells ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Osteogenic Potential ◽  
Donor Age

The relationships between donor age and gender and initial isolation yield and the osteogenic potentials of human bone marrow stromal cells (hBMSCs) have not been clearly elucidated. The authors investigated whether isolation yields and the osteogenic differentiation potentials of hBMSCs are indeed dependent on donor age or gender. Fresh bone marrow was aspirated from iliac crest of 72 donors (mean age 54.1 years; range, 23-84 years; 39 men and 33 women) undergoing total hip arthroplasty. Numbers of mononuclear cells, numbers of colony forming unit-fibroblasts (CFU-Fs) and alkaline phosphatase (ALP)-positive CFU-Fs, and numbers of BMSCs after isolation culture were not found to be significantly dependent on donor age or gender. Moreover, no significant age- or gender-related differences were observed in terms of the proliferation activities, ALP activities, and calcium contents of BMSCs during in vitro osteogenic differentiation. The data obtained from 72 human donors revealed no significant age- or genderrelated differences among hBMSCs in terms of isolation yields, proliferation activities, and osteogenic potentials.

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

Blood ◽  
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.

scholarly journals Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells

Theranostics ◽  
2018 ◽  
Vol 8 (5) ◽  
pp. 1421-1434 ◽  
Author(s):  
Michaela Oeller ◽  
Sandra Laner-Plamberger ◽  
Sarah Hochmann ◽  
Nina Ketterl ◽  
Martina Feichtner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tissue Factor ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Cell Transplants ◽  
Novel Strategy ◽  
Selection Of

An Alternative Growth Factor for Hematopoietic Stem Cells and Megakaryocytopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2060-2060 ◽  
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.

GLI2 Transcription Factor Modulates CD40 Ligand Expression In Bone Marrow Stromal Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4271-4271
Author(s):  
Matthew S. Neil ◽  
Joel R. Dennison ◽  
Martin E. Fernandez-Zapico ◽  
Sherine F. Elsawa
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Stromal Cells ◽  
Cell Biology ◽  
Bone Marrow Stromal Cells ◽  
Cd40 Ligand ◽  
Malignant Cell ◽  
Marrow Stromal Cells ◽  
Rt Pcr

Abstract The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells within the host. Cytokines play a key role in mediating this crosstalk between malignant cells and the tumor microenvironment. Although this interaction is clearly established, many of the molecular mechanisms mediating these signaling events remain elusive. In previous work, we have shown that the cytokine CCL5 can regulate the expression and activity of the transcription factor GLI2 in stromal cells. GLI2 is an effector of hedgehog (HH) signaling; however, our studies reveal a role for GLI2 independent of HH. GLI2, in turn, increases the expression and secretion of IL-6 in the tumor microenvironment, highlighting a novel role for GLI2 in directly modulating cytokine genes. We therefore screened for additional cytokines regulated by this axis in HS-5 bone marrow stromal cells using expression arrays. We found that GLI2 can modulate the expression of several cytokines including CD40 ligand (CD40L), CCL2, CCL7 and CXCL11. CD40L is an immunomodulatory and proinflammatory molecule that has been shown to play a role in multiple hematologic malignancies including chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and acute myeloid leukemia (AML) pathogenesis. We first characterized the regulation of CD40L, because of the importance of CD40L in malignant cell biology. Gene expression results were validated by RT-PCR in two additional bone marrow stromal cell lines Saka and L88 where overexpression of GLI2 resulted in increased CD40L expression. Furthermore, we detected increased levels of soluble CD40L (sCD40L) in L88 and Saka cells overexpressing GLI2. Conversely, knockdown of GLI2 resulted in reduced expression and secretion of this cytokine. Biochemical mapping of the domains critical for this GLI2 regulatory mechanism reveals that N-terminal transcriptional domain is dispensable, as a mutant form of GLI2 lacking this domain (delta N-GLI2) increases CD40L gene expression by RT-PCR, protein expression by western blot and sCD40L secretion by ELISA, beyond that of the full-length GLI2 levels. Bioinformatics analysis of the CD40L promoter indicates the presence of 3 candidate binding sites for GLI proteins, suggesting the modulation of CD40L by GLI2 is mediated by direct interaction with its promoter. Ongoing experiments are aimed at determining the mechanism by which GLI2 regulates CD40L expression (either by direct binding to the CD40L promoter or through another indirect pathway). Together, our data identify a novel mechanism controlling CD40L in stromal cells in the bone marrow microenvironment. Because of the importance of CD40L in malignant cell biology, understanding the mechanism of its regulation is of great importance for the development of therapies aimed at targeting the malignant bone marrow tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age

2002 ◽  
Vol 8 (6) ◽  
pp. 911-920 ◽  
Author(s):  
S.C. Mendes ◽  
J.M. Tibbe ◽  
M. Veenhof ◽  
K. Bakker ◽  
S. Both ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Tissue ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Culture Conditions ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Donor Age
Sign in / Sign up

Export Citation Format

Share Document