scholarly journals Comparative analysis of gene transcripts for cell signaling receptors in bone marrow-derived hematopoietic stem/progenitor cell and mesenchymal stromal cell populations

2013 ◽  
Vol 4 (5) ◽  
pp. 112 ◽  
Author(s):  
Khairul Anam ◽  
Thomas A Davis
Keyword(s):  
Bone Marrow ◽  
Comparative Analysis ◽  
Cell Signaling ◽  
Mesenchymal Stromal Cell ◽  
Progenitor Cell ◽  
Stromal Cell ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Gene Transcripts ◽  
Signaling Receptors

Loss of TGF-β Signaling in Bone Marrow Mesenchymal Progenitors Promotes Adipocyte over Osteoblast Differentiation but Does Not Disrupt the HSC Niche

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 666-666
Author(s):  
Grazia Abou Ezzi ◽  
Teerawit Suparkorndej ◽  
Bryan Anthony ◽  
Jingzhu Zhang ◽  
Shilpi Ganguly ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mesenchymal Stromal Cell ◽  
Stromal Cell ◽  
Transforming Growth Factor ◽  
Cell Populations ◽  
Mesenchymal Progenitors ◽  
Hsc Niche

Abstract Hematopoietic stem cells (HSCs) reside in specialized microenvironments (niches) in the bone marrow. Several mesenchymal stromal cells have been implicated in hematopoietic niches, including osteoblasts, pericytes, CXCL12-abundant reticular (CAR) cells, and mesenchymal stem cells (MSCs). Members of the transforming growth factor (TGF) superfamily, in particular TGF-β, have a well-documented role in regulating osteoblast development. However, the contribution of TGF family member signaling to the establishment and maintenance of hematopoietic niches is largely unknown. Here, we characterize the role of transforming growth factor-β (TGF-β) signaling in mesenchymal stromal cells on the HSC niche. TGF-β receptor 2 (encoded by Tgfbr2) is required for all TGF-β signaling. To selectively disrupt TGF-β signaling in bone marrow mesenchymal stromal cells, we generated Osx-C re Tgfbr2fl/fl mice. Osx-Cre targets most bone marrow mesenchymal stromal cells (including osteoblasts, CAR cells, MSCs, pericytes, and adipocytes) but not endothelial cells or hematopoietic cells. Osx-C re Tgfbr2fl/fl mice are severely runted and most die by 4 weeks of age. We analyzed mice at 3 weeks, when the mice appeared healthy. Osteoblast number was severely reduced in Osx-C re Tgfbr2fl/fl mice, as assessed by histomorphometry and immunostaining for osteocalcin. Accordingly, microCT analysis demonstrated reduced tissue mineral density and cortical thickness of long bone and marked trabecularization of long bones in diaphyseal regions. Surprisingly, marrow adiposity, as measured by osmium tetroxide staining with microCT, was strikingly increased in Osx-C re Tgfbr2fl/fl mice. CAR cells are mesenchymal progenitors with osteogenic and adipogenic potential in vitro. To assess CAR cells, we generated Osx-Cre Tgfrb2fl/fl x Cxcl12gfp mice. Surprisingly, CAR cell number was significantly increased. However, despite the increase in CAR cells, the number of CFU-osteoblast (CFU-OB) in Osx-C re Tgfbr2fl/fl mice is nearly undetectable. Together, these data suggest that TGF-b signaling contributes to lineage commitment of mesenchymal progenitors. Specifically, our data suggest that TGF-β signaling suppresses commitment to the osteoblast lineage, while increasing adipogenic differentiation. We next asked whether alterations in bone marrow stromal cells present in Osx-C re Tgfbr2fl/fl mice affect HSC number or function. The increase in marrow adipocytes and loss of osteolineage cells is predicted to impair HSC maintenance, while the increase in CAR cells might augment HSCs. Osx-Cre Tgfrb2fl/fl mice have modest leukopenia, but normal red blood cell and platelet counts. Bone marrow and spleen cellularity are reduced, even after normalizing for body weight. The frequency of phenotypic HSCs (defined as Kit+ lineage- Sca+ CD34- Flk2- cells) is comparable to control mice. To assess HSC function, we performed competitive repopulation assays with bone marrow from Osx-Cre Tgfrb2fl/fl or control mice. Surprisingly, these data show that the long-term multi-lineage repopulating activity of HSCs from Osx-Cre Tgfrb2fl/fl mice is normal. Moreover, serial transplantation studies suggest that the self-renewal capacity of HSCs is normal. Thus, despite major alterations in mesenchymal stromal cell populations, the HSC niche is intact in Osx-Cre Tgfrb2fl/fl mice. Collectively, these data show that TGF-b signaling in mesenchymal progenitors is required for the proper development of multiple stromal cell populations that contribute to hematopoietic niches. Studies are underway to assess the impact of post-natal deletion of Tgfbr2 in mesenchymal stromal cell on hematopoietic niches. Since drugs that modulate the activity of TGF-b are in development, this research may suggest novel approaches to modulate hematopoietic niches for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.

Regulation of β1-Integrin by Mir-134 in Mesenchymal Stromal Cells – Implications for Mesenchymal Stromal Cell Adherence and Hematopoietic Stem Cell Interaction

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3459-3459
Author(s):  
Friedrich Stölzel ◽  
David M. Poitz ◽  
Laleh S. Arabanian ◽  
Jens Friedrichs ◽  
Denitsa Docheva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stromal Cell ◽  
Stromal Cell ◽  
Luciferase Activity ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Β1 Integrin ◽  
Hematopoietic Stem ◽  
Transfected Cells

Abstract Abstract 3459 The different intra- and extracellular constituents of the hematopoietic stem cell (HSC) niche in the human bone marrow are tightly regulated and of momentous importance for various properties of HSCs. Some of these are regulated through β1-Integrins (CD29) which therefore dramatically influence HSC and mesenchymal stromal cell (MSC) interaction in the niche. Important regulators within these cells are microRNAs (miRNAs). These small, non-coding RNAs control the expression of around two-thirds of the human protein-coding genes. One of these miRNAs, miR-134, previously referred to be a “brain-specific” miRNA was shown to be highly expressed in MSCs in tissue-studies conducted by our group. Since the central nervous system was recently shown to be closely connected to the regulation of HSCs and MSCs, we asked whether miR-134 which has several conserved binding seed-match sequences within the 3'UTR of β1-Integrin, regulates MSC mediated properties in the bone marrow niche. Screening of human MSC cell lines (n=4) by western blotting revealed highest β1-Integrin expression in SCP-1 cells. Transfection of SCP-1 with either siRNA directed against β1-Integrin (siCD29) or pre-miRNA-134 (pre134) revealed a downregulation of β1-Integrin at the mRNA level only in siRNA transfected cells, p=0.01. In contrast, at the protein level, as measured by western blot and FACS analysis, p=0.002, β1-Integrin was downregulated by siCD29 as well as by pre134, indicating a miRNA-specific action of repression. Confirmatory, the 3'UTR of β1-Integrin, which contains several putative binding sites for miR-134, was cloned into a pMiRReporter vector and luciferase activity was measured after cotransfection with pre134. The luciferase activity was significantly reduced in pre134 transfected cells [1.80 ± 0.46 (preCo) vs. 0.99 ± 0.49 (pre134); p<0.001]. To evaluate whether pre134 mediated reduction of β1-Integrin can modulate the adhesion potential of SCP-1, atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) was performed. Indeed, transfection of SCP-1 with siCD29 or pre134 resulted in a significantly reduced adherence as compared to their respective controls, p<0.001 and p<0.01. Furthermore, using AFM-based SCFS we investigated the interaction between 32D-cells, which have a high surface expression of the natural interaction partner of β1-Integrin VCAM-1, and SCP-1 cells. Here again, we were able to show, that 32D show a significantly lower adhesion potential to siCD29 and pre134-transfected SCP-1, p<0.001 and p<0.001, respectively. In a translational approach MSCs from healthy bone marrow donors (n=30) and from MDS patients (n=17) were screened for miRNA-expression. This analysis revealed 50% higher miR-134 transcript levels in MSCs from MDS patients [0.0057 ± 0.0021 (healthy) vs. 0.0127 ± 0.0045 (MDS); p<0.001], suggesting a potential role of this miRNA in regulating its MSC adhesion. Regulation of adhesion of MSCs and to MSCs is important for various components of the bone marrow niche. Here, we demonstrate for the first time that β1-Integrin mediated adhesion of MSCs themselves and other cell types onto MSCs via β1-Integrin receptors can be inhibited via miR-134 overexpression. Furthermore, this newly characterized mechanism provides evidence for a potential anti-adhesive influence of miR-134. While this might not only influence adhesion, other mechanisms such as homing of HSCs as well as other cell types, might be affected by modification of miR-134 expression in the stromal niche. Disclosures: Platzbecker: Amgen: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2401-2401
Author(s):  
Jingzhu Zhang ◽  
Daniel C. Link
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Sympathetic Nerves ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Lineage Mapping

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice. Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells. There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes. Disclosures No relevant conflicts of interest to declare.

Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5544-5544
Author(s):  
Mariluz P. Mojica-Henshaw ◽  
L. Jeanne Pierce ◽  
John D. Phillips ◽  
Vicente Planelles ◽  
Gerald J. Spangrude
Keyword(s):  
Bone Marrow ◽  
Transgene Expression ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Random Sequence ◽  
Transduction Efficiency ◽  
Cell Populations ◽  
Lentivirus Vector ◽  
Cmv Promoter ◽  
Hematopoietic Stem

Abstract We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

Physiologic Hematopoiesis Is Dependent on Stromal Expression of Laminin-γ1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3596-3596
Author(s):  
Sreemanti Basu ◽  
Irene Hernandez ◽  
Mark Zogg ◽  
Hartmut Weiler ◽  
Karen-Sue B. Carlson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Progenitor Cells ◽  
Stromal Cell ◽  
Hematopoietic Cell ◽  
Cell Populations ◽  
Gene Recombination ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Hematopoietic Niche

Abstract OBJECTIVE: The microenvironment of the bone marrow hematopoietic niche includes: 1) blood vessels; 2) adjacent stromal cells; 3) hematopoietic cells; and 4) cytokines, growth factors, and structural support molecules. Injury to the bone marrow niche affects hematopoietic cells through loss of either physical contact with stromal cells or niche-derived growth factors. Extracellular matrix factors required to maintain the steady-state bone marrow perivascular niche and hematopoiesis have not been fully identified. Here, we examine the role of the extracellular matrix protein laminin-γ1 in adult bone marrow for maintaining the perivascular hematopoietic niche and hematopoiesis. METHODS: A global and inducible laminin-γ1 deficient mouse, hereafter referred to as mutant, was generated in which LAMC1 gene recombination could be monitored by a fluorescent reporter transgene. Tamoxifen was used to induce LAMC1 gene recombination and knock-down of laminin-γ1 protein expression in 8-12 week old mice. Tamoxifen-treated mice lacking the inducible Cre transgene were used as controls for all experiments. Analysis of mutant mice was performed 17-24 days following the first dose of tamoxifen. Bone marrow samples were examined by immunohistochemistry for the presence of laminin-1 protein, and hematopoietic tissues, including bone marrow, peripheral blood, spleen, thymus and lymph node were analyzed by flow cytometry for hematopoietic stem and progenitor cells, and mature hematopoietic cell populations, and by ex vivo hematopoietic progenitor cell colony forming assays. To examine the stromal cell contribution to laminin-γ1 mediated hematopoietic dysfunction, control and mutant mice that had not yet undergone LAMC1 gene recombination were then transplanted with wild type bone marrow. After full hematopoietic reconstitution, chimeric mice were induced with tamoxifen and hematopoietic stem and progenitor cells and mature hematopoietic cell populations were examined. RESULTS: LAMC1 gene recombination in the bone marrow of tamoxifen-treated mutant animals varied between 20-45%. With LAMC1 gene recombination, laminin-protein in the bone marrow is rapidly depleted. Bone marrow blood vessels dilate, and the bone marrow becomes hypocellular. Hematopoietic stem and progenitor cells are reduced in number, as are bone marrow B-cell progenitors, and thymic double-positive T-cells. Erythrocyte and thrombocyte numbers are not changed, nor were NK cell populations, the majority of myeloid cell subtypes, or mature B cells in the bone marrow and blood. CD8+ T-cells were increased within the bone marrow, as were CD11b+, Ly6Clo Ly6G+ myeloid cells in the peripheral blood. Examination of gene recombination within each of these cell types was not consistent with cell-intrinsic mechanism of hematopoietic alterations. Analysis of bone marrow chimeric animals identified loss of stromal cell-produced laminin-γ1 as a significant mediator of these hematopoietic alterations. CONCLUSIONS: The rapid depletion of laminin-γ1 from the bone marrow indicates a high basal turnover rate for the extracellular matrix in the bone marrow. The attendant hematopoietic dysfunction that follows loss of laminin-γ1 production in the bone marrow is specific for immature hematopoietic cell populations, and is dependent on stromal cell production of laminin-γ1. This study identifies laminin-γ1 as a niche-dependent regulator of hematopoietic stem and progenitor cell populations, and suggests that that regulation of its production and degradation may be new targets for the study of the hematopoietic niche. Disclosures No relevant conflicts of interest to declare.

scholarly journals Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 473-481 ◽  
Author(s):  
AE Gibbons ◽  
P Price ◽  
GR Shellam
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cell ◽  
Murine Cytomegalovirus ◽  
Cell Populations ◽  
Mcmv Infection ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Marrow Cells

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony- forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst- forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV- infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

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